Quaternary ammonium compounds useful as muscarinic receptor antagonists

ABSTRACT

The invention provides compounds of the formula: 
                         
in salt or zwitterionic form or a pharmaceutically acceptable salt thereof, wherein R 1-6 , a, Z and Q are as defined in the specification. These compounds are muscarinic receptor antagonists. The invention also provides pharmaceutical compositions containing such compounds, processes for preparing such compounds and methods of using such compounds to, for example, treat pulmonary disorders such as chronic obstructive pulmonary disease and asthma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.13/473,792, filed May 17, 2012, now allowed, which is a divisionalapplication of U.S. Ser. No. 12/148,887, filed Apr. 23, 2008, now U.S.Pat. No. 8,202,891, which claims the benefit of U.S. ProvisionalApplication No. 60/925,951, filed on Apr. 24, 2007; the entiredisclosures of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to quaternary ammonium compounds havingmuscarinic receptor antagonist or anticholinergic activity. Theinvention also relates to pharmaceutical compositions comprising thesecompounds, processes for preparing them and methods of use to treatpulmonary disorders.

2. State of the Art

Pulmonary or respiratory disorders, such as chronic obstructivepulmonary disease (COPD) and asthma, afflict many millions of peopleworldwide and such disorders are a leading cause of morbidity andmortality. Muscarinic receptor antagonists are known to providebronchoprotective effects and therefore, such compounds are useful fortreating respiratory disorders, such as COPD and asthma. When used totreat such disorders, muscarinic receptor antagonists are typicallyadministered by inhalation. However, even when administered byinhalation, a significant amount of the muscarinic receptor antagonistis often absorbed into the systemic circulation resulting in systemicside effects, such as dry mouth, mydriasis and cardiovascular sideeffects. Additionally, many inhaled muscarinic receptor antagonists havea relatively short duration of action requiring that they beadministered several times per day. Such a multiple-daily dosing regimeis not only inconvenient but also creates a significant risk ofinadequate treatment due to patient non-compliance with the requiredfrequent dosing schedule.

Accordingly, a need exists for new muscarinic receptor antagonists. Inparticular, a need exists for muscarinic receptor antagonists havinghigh potency, reduced systemic side effects when administered byinhalation, and a long duration of action thereby allowing foronce-daily or even once-weekly dosing. In addition, a need exists formuscarinic receptor antagonists having high affinity for the receptorand a long receptor half life. Such compounds are expected to beparticularly effective for treating pulmonary disorders, such as COPDand asthma, while reducing or eliminating side effects, such asdry-mouth and constipation.

SUMMARY OF THE INVENTION

The present invention provides novel quaternary ammonium compounds whichhave muscarinic receptor antagonist or anticholinergic activity. Amongother properties, compounds of this invention have been found to possessimproved binding affinity for hM₂ and hM₃ muscarinic receptor subtypes,have longer receptor half-lives, have a larger therapeutic window, orhave greater potency compared to related compounds. Accordingly,compounds of the invention are expected to be useful and advantageous astherapeutic agents for treating pulmonary disorders.

One aspect of the invention relates to compounds having formula I:

in salt or zwitterionic form, wherein:

R¹ is selected from —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl,—C₁₋₃alkylene-SCH₃, —C₃₋₉cycloalkyl, and heteroaryl; R² is an aryl orheteroaryl group; R³ is selected from H and —C₀₋₁alkylene-OH; or when R¹is —C₃₋₉cycloalkyl, R³ can form a double bond with the carbon atom onthe —C₃₋₉cycloalkyl group; or —CR¹R² together form a group of formula:

a is 0 or an integer of from 1 to 3; each R⁴ is independently selectedfrom fluoro and —C₁₋₄alkyl;

R⁵ is selected from —C₁₋₅alkyl and —C₀₋₁alkyleneC₃₋₅cycloalkyl;

R⁶ is selected from —C₁₋₃alkyl, —C₁₋₂alkyleneC₃₋₇cycloalkyl,—C₀₋₄alkylene-OH, —C₁₋₂alkylene-C(O)O—C₁₋₄alkyl, and—C₁₋₂alkylene-C(O)NR^(6a)R^(6b); where R^(6a) and R^(6b) areindependently selected from H and —C₁₋₄alkyl; or R⁶ is taken with R⁵ toform —C₃₋₅alkylene-;

Z is selected from a bond, —O—, —S—, —S(O)—, —SO₂—, —SO₂—NR^(Z1)—,—NR^(Z1)—SO₂—, —C(O)—, —OC(O)—, —C(O)O—, —NR^(Z1)C(O)—, —C(O)NR^(Z1)—,—NR^(Z2)—C(O)—NR^(Z3)—, —NR^(Z2)—C(S)—NR^(Z3)—, —CH(OH)—, and—C(═N—O—R^(Z4))—; where R^(Z1) is selected from H and —C₁₋₄alkyl; R^(Z2)and R^(Z3) are independently selected from H, —C₁₋₄alkyl, and—C₃₋₆cycloalkyl, or R^(Z2) and R^(Z3) are taken together to form—C₂₋₄alkylene- or —C₂₋₃alkenylene-; and R^(Z4) is selected from—C₁₋₄alkyl and benzyl; and

Q is an aryl or heteroaryl group;

wherein the —C₃₋₉cycloalkyl in R¹ and the aryl in R² are optionallysubstituted with 1 to 5 R groups independently selected from —C₁₋₄alkyl,—C₂₋₄alkenyl, —C₂₋₄alkynyl, —C₃₋₆cycloalkyl, cyano, halo, —OR^(a),—SR^(a), —S(O)R^(a), —S(O)₂R^(a), and —NR^(b)R^(c); where each R^(a) isindependently selected from H, —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl,and —C₃₋₆cycloalkyl; and each R^(b) and R^(c) is independently selectedfrom H, —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, and —C₃₋₆cycloalkyl;wherein the aryl in Q is optionally substituted with 1 to 5 R^(Q) groupsindependently selected from halo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano,—C₀₋₂alkylene-COOH, —C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl, —S—C₁₋₄alkyl,—NH—C(O)—C₁₋₄alkyl, —N-di-C₁₋₄alkyl, and —N⁺(O)O; wherein each alkyl,alkylene, alkenyl, alkenylene, alkynyl, and cycloalkyl group in R, R³⁻⁶,Z, Q, and R^(Q), is optionally substituted with 1 to 5 fluoro atoms; andeach —CH₂— group in —(CH₂)₁₋₄— is optionally substituted with 1 or 2substituents independently selected from —C₁₋₂alkyl, —OH, fluoro, andphenyl; and pharmaceutically acceptable salts thereof.

Another aspect of the invention relates to quaternary ammonium compoundshaving formula II:

and pharmaceutically acceptable salts thereof, where X⁻ is an anion of apharmaceutically acceptable acid; and R¹⁻⁶, a, Z and Q are as definedfor formula I.

Among the compounds of formula I, compounds of particular interest arethose having an inhibition dissociation constant (K_(i)) for binding tothe M₃ receptor subtype of less than or equal to 100 nM; in particularhaving a K_(i) less than or equal to 50 nM; more particularly having aK_(i) less than or equal to 10 nM; and even more particularly having aK_(i) less than or equal to 1.0 nM.

Another aspect of the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a compound of theinvention. Such compositions may optionally contain other therapeuticagents such as steroidal anti-inflammatory agents (e.g.,corticosteroids), β₂ adrenergic receptor agonists, phosphodiesterase-4inhibitors, and combinations thereof. Accordingly, in yet another aspectof the invention, a pharmaceutical composition comprises a compound ofthe invention, a second active agent, and a pharmaceutically acceptablecarrier. Another aspect of the invention pertains to a combination ofactive agents, comprising a compound of the invention and a secondactive agent. The compound of the invention can be formulated togetheror separately from the additional agent(s). When formulated separately,a pharmaceutically acceptable carrier may be included with theadditional agent(s). Thus, yet another aspect of the invention relatesto a combination of pharmaceutical compositions, the combinationcomprising: a first pharmaceutical composition comprising a compound ofthe invention and a first pharmaceutically acceptable carrier; and asecond pharmaceutical composition comprising a second active agent and asecond pharmaceutically acceptable carrier. This invention also relatesto a kit containing such pharmaceutical compositions, for example wherethe first and second pharmaceutical compositions are separatepharmaceutical compositions.

Compounds of the invention possess muscarinic receptor antagonistactivity, and are therefore expected to be useful as therapeutic agentsfor treating patients suffering from a disease or disorder that istreated by blocking the muscarinic receptor. Thus, one aspect of theinvention is directed to a method of producing bronchodilation in apatient, comprising administering to the patient abronchodilation-producing amount of a compound of the invention. Theinvention is also directed to method of treating a pulmonary disordersuch as chronic obstructive pulmonary disease or asthma, comprisingadministering to a patient a therapeutically effective amount of acompound of the invention. Another aspect of the invention relates to amethod for antagonizing a muscarinic receptor in a mammal comprisingadministering to the mammal, a muscarinic receptor-antagonizing amountof a compound of the invention.

Since compounds of the invention possess muscarinic receptor antagonistactivity, such compounds are also useful as research tools. Accordingly,one aspect of the invention pertains to a method of using a compound ofthe invention as a research tool, the method comprising conducting abiological assay using a compound of the invention. Compounds of theinvention can also be used to evaluate new chemical compounds. Thusanother aspect of the invention relates to a method of evaluating a testcompound in a biological assay, comprising: (a) conducting a biologicalassay with a test compound to provide a first assay value; (b)conducting the biological assay with a compound of the invention toprovide a second assay value; wherein step (a) is conducted eitherbefore, after or concurrently with step (b); and (c) comparing the firstassay value from step (a) with the second assay value from step (b).Exemplary biological assays include a muscarinic receptor binding assayand a bronchoprotection assay in a mammal. Still another aspect of theinvention is directed to a method of studying a biological system orsample comprising a muscarinic receptor, the method comprising: (a)contacting the biological system or sample with a compound of theinvention; and (b) determining the effects caused by the compound on thebiological system or sample.

The invention is also directed to processes and intermediates useful forpreparing compounds of the invention. Accordingly, another aspect of theinvention relates to a process of preparing compounds of the invention,comprising: (a) reacting a compound of formula 1 with a compound offormula 2 or reacting a compound of formula 1′ with a compound offormula 2′ to produce a compound of formula 3, and reacting the compoundof formula 3 with an organic substrate containing an R⁶ group; or (b)reacting a compound of formula 4 with a compound of formula 2; or (c)reacting a compound of formula 4 with a compound of formula 5 to producea compound of formula 6, and reacting the compound of formula 6 with acompound of formula 7; and recovering the product in salt orzwitterionic form, to provide a compound of formula I; wherein compoundsof formula 1, 1′, 2, 2′, and 3-7 are as defined herein. In otheraspects, the invention is directed to products prepared by any of theprocesses described herein.

Yet another aspect of the invention is directed to the use of a compoundof the invention for the manufacture of a medicament, especially for themanufacture of a medicament useful for treating a pulmonary disorder(such as chronic obstructive pulmonary disease and asthma), forproducing bronchodilation, or for antagonizing a muscarinic receptor ina mammal. Still another aspect of the invention pertains to the use of acompound of the invention as a research tool. Other aspects andembodiments of the invention are disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to compounds having formula I:

in salt or zwitterionic form, or a pharmaceutically acceptable saltthereof. More specifically, the invention is directed to quaternaryammonium compounds having formula II:

or a pharmaceutically acceptable salt thereof, where X⁻ is an anion of apharmaceutically acceptable acid. The term “quaternary ammoniumcompound” refers to a compound that is derived from ammonium hydroxideor from an ammonium salt, wherein all four hydrogen atoms of the NH₄ ⁻ion have been replaced by organic groups.

As used herein, the term “compound of the invention” is intended toinclude compounds of formula I as well as the species embodied informulas such as II, IIa, IIb, IIc, IId, IIe, and IIf. The compounds ofthe invention are quaternary ammonium salts and may be converted betweendifferent salt forms using state of the art methodologies, for example,using ion exchange chromatography. Also, the compounds can be obtainedin the form of solvates, and such solvates are included within the scopeof this invention. Accordingly, those skilled in the art will recognizethat reference to a compound herein, for example, reference to a“compound of the invention” includes reference to a compound of formulaI as well as to any pharmaceutically acceptable salt forms andpharmaceutically acceptable solvates of that compound unless otherwiseindicated.

The compounds of the invention may contain one or more chiral centersand so may exist in a number of stereoisomeric forms. When such chiralcenters are present, this invention is directed to racemic mixtures,pure stereoisomers (i.e., enantiomers or diastereomers),stereoisomer-enriched mixtures, and the like unless otherwise indicated.When a chemical structure is depicted without any stereochemistry, it isunderstood that all possible stereoisomers are encompassed by suchstructure. Thus, for example, the term “compound of formula I” isintended to include all possible stereoisomers of the compound.Similarly, when a particular stereoisomer is shown or named herein, itwill be understood by those skilled in the art that minor amounts ofother stereoisomers may be present in the compositions of this inventionunless otherwise indicated, provided that the utility of the compositionas a whole is not eliminated by the presence of such other isomers.Individual enantiomers may be obtained by numerous methods that are wellknown in the art, including chiral chromatography using a suitablechiral stationary phase or support, or by chemically converting theminto diastereomers, separating the diasteromers by conventional meanssuch as chromatography or recrystallization, then regenerating theoriginal enantiomers. Additionally, where applicable, all cis-trans orE/Z isomers (geometric isomers), tautomeric forms and topoisomeric formsof the compounds of this invention are included within the scope of thisinvention unless otherwise specified.

In particular, the compounds of formula I contain a chiral center at thecarbon atom indicated by the symbol * in the following partial formula(shown without optional substituents for clarity):

In one embodiment of this invention, the carbon atom identified by thesymbol * has the (R) configuration. In this embodiment, compounds offormula I have the (R) configuration at the carbon atom identified bythe symbol * or are enriched in a stereoisomeric form having the (R)configuration at this carbon atom. In another embodiment, the carbonatom identified by the symbol * has the (S) configuration. In thisembodiment, compounds of formula I have the (S) configuration at thecarbon atom identified by the symbol * or are enriched in astereoisomeric form having the (S) configuration at this carbon atom.

The compounds of the invention, as well as those compounds used in theirsynthesis, may also include isotopically-labeled compounds, i.e., whereone or more atoms have been enriched with atoms having an atomic massdifferent from the atomic mass predominately found in nature. Examplesof isotopes that may be incorporated into the compounds of formula I,for example, include, but are not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸Oand ¹⁷O.

The compounds of the invention have been found to possess muscarinicreceptor antagonist activity. Among other properties, compounds of theinvention have been found to possess improved binding affinity for hM₂and hM₃ muscarinic receptor subtypes, have longer receptor half-lives,and have greater potency compared to related compounds, and are expectedto be useful as therapeutic agents for treating pulmonary disorders.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially-available AutoNom software (MDL, San Leandro,Calif.).

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of theinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of the invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from the invention unless specifically indicated.

R¹ is selected from —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl,—C₁₋₃alkylene-SCH₃, —C₃₋₉cycloalkyl, and heteroaryl. In one embodiment,R¹ is —C₁₋₆alkyl, such as —CH₂CH(CH₃)₂. In another embodiment, R¹ is—C₂₋₆alkenyl, such as —CH₂CH═CH₂. In still another embodiment, R¹ is—C₂₋₆alkynyl, such as —C≡CH. In still another embodiment, R¹ is—C₁₋₃alkylene-SCH₃, such as —(CH₂)₂SCH₃. In still another embodiment, R¹is —C₃₋₉cycloalkyl, such as cyclopropyl and cyclopentyl. The—C₃₋₉cycloalkyl group is optionally substituted with 1 to 5 R groupsindependently selected from —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl,—C₃₋₆cycloalkyl, cyano, halo, —OR^(a), —SR^(a), —S(O)R^(a), —S(O)₂R^(a),and —NR^(b)R^(c); where each R^(a) is independently selected from H,—C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, and —C₃₋₆cycloalkyl; and eachR^(b) and R^(c) is independently selected from H, —C₁₋₄alkyl,—C₂₋₄alkenyl, —C₂₋₄alkynyl, and —C₃₋₆cycloalkyl. In one embodiment, the—C₃₋₉cycloalkyl group is unsubstituted. When present, each R group maybe at any position of the —C₃₋₉cycloalkyl ring to which it is attached.When more than one R substituent is present, i.e., a is 2, 3, 4 or 5,the substituents can be on the same or on different carbon atoms. In oneembodiment, R is independently selected from —C₁₋₄alkyl (e.g. methyl,ethyl, n-propyl, isopropyl), halo (e.g., fluoro or chloro) and —OR^(1a)(e.g., hydroxy, methoxy, ethoxy). Each of the aforementioned alkyl,alkenyl, alkynyl, and cycloalkyl groups in R may be substituted with 1to 5 fluoro atoms. It is understood that when referring to these groupsin R, reference is also made to any such groups that might be present inthe R^(a), R^(b), and R^(c) moieties. For example, R¹ can be —C₁₋₄alkylsuch as difluoromethyl, trifluoromethyl, and 2,2,2-trifluoroethyl, or—OR^(a), where R^(a) is difluoromethyl or trifluoromethyl.

In another embodiment, R¹ is heteroaryl, such as thiophene (including2-thiophene and 3-thiophene).

R² is an aryl or heteroaryl group. In one particular embodiment, R² isan aryl group such as phenyl or naphthalenyl; in another embodiment Aris phenyl. In one particular embodiment, R² is a heteroaryl group suchas thiophene (including 2-thiophene and 3-thiophene). The aryl in R² maybe substituted with 1 to 5 R groups independently selected from—C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, —C₃₋₆cycloalkyl, cyano, halo,—OR^(a), —SR^(a), —S(O)R^(a), —S(O)₂R^(a), and —NR^(b)R^(c). Each R^(a)is independently selected from H, —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyland —C₃₋₆cycloalkyl. Each R^(b) and R^(c) is independently selected fromH, —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, and —C₃₋₆cycloalkyl. Each ofthe aforementioned alkyl, alkenyl, alkynyl, and cycloalkyl groups in Rmay be substituted with 1 to 5 fluoro atoms. It is understood that whenreferring to these groups in R², reference is also made to any suchgroups that might be present in the R^(a), R^(b), and R^(c) moieties. Inone particular embodiment, R² is phenyl and is unsubstituted. In anotherembodiment, R² is phenyl and is substituted with 1 or 2 R groupsindependently selected from —C₁₋₄alkyl, (for example, —CH₃), halo (forexample fluoro) and —OR^(a) (where R^(a) is H or —C₁₋₄alkyl such as—CH₃). In one particular embodiment, R² is phenyl and is substitutedwith 1 fluoro atom.

R³ is selected from H and —C₀₋₁alkylene-OH, where the alkylene group isoptionally substituted with 1 to 5 fluoro atoms. In one embodiment, R³is selected from H and —OH. In another embodiment, R³ is —OH. In yetanother embodiment, R³ is —CH₂OH. Alternatively, when R¹ is—C₃₋₉cycloalkyl, R³ may form a double bond with the carbon atom on the—C₃₋₉cycloalkyl group, an embodiment which may be depicted as:

In one particular embodiment, R¹ is cyclopentyl and R³ forms a doublebond with the carbon atom on the cyclopentyl group.

In yet another embodiment, —CR¹R² together form a group of formula:

In one such embodiment, R³ is H.

The nitrogen containing ring can have from 3 to 6 carbon atoms in thering, and thus is depicted as:

This depiction is intended to include the following nitrogen containingrings:

In one particular embodiment, the nitrogen containing ring is:

The values for a are 0, 1, 2 or 3; and even more particularly 0 or 1. Inone embodiment, a is 0.

When present, each R⁴ is independently selected from fluoro and—C₁₋₄alkyl. When more than one R⁴ substituent is present, i.e., a is 2or 3, the substituents can be on the same or on different carbon atoms.Exemplary R⁴ groups include, but are not limited to, methyl, ethyl, andfluoro. The alkyl group in R⁴ may be substituted with 1 to 5 fluoroatoms. For example, R⁴ can be difluoromethyl or trifluoromethyl.

R⁵ is selected from —C₁₋₅alkyl and —C₀₋₁alkyleneC₃₋₅cycloalkyl. Each ofthe aforementioned alkyl, alkylene, and cycloalkyl groups in R⁵ may besubstituted with 1 to 5 fluoro atoms. In one embodiment, R⁵ is—C₁₋₅alkyl such as —CH₃.

R⁶ is selected from —C₁₋₃alkyl, —C₁₋₂alkyleneC₃₋₇cycloalkyl,—C₀₋₄alkylene-OH, —C₁₋₂alkylene-C(O)O—C₁₋₄alkyl, and—C₁₋₂alkylene-C(O)NR^(6a)R^(6b). R^(6a) and R^(6b) are independentlyselected from H and —C₁₋₄alkyl. Exemplary —C₁₋₃alkyl groups include—CH₃, —CH₂CH₃, and —(CH₂)₂CH₃. Exemplary —C₀₋₂alkyleneC₃₋₇cycloalkylgroups include —CH₂-cyclopropyl. Exemplary —C₀₋₄alkylene-OH groupsinclude —(CH₂)₂OH. Exemplary —C₁₋₂alkylene-C(O)O—C₁₋₄alkyl groupsinclude —CH₂—C(O)OCH₃. Exemplary —C₁₋₂alkylene-C(O)NR^(6a)R^(6b) groupsinclude —CH₂—C(O)NH₂. Alternately, R⁶ is taken with R⁵ to form—C₃₋₅alkylene-. Each of the aforementioned alkyl, alkylene, andcycloalkyl groups in R⁶ may be substituted with 1 to 5 fluoro atoms. Itis understood that when referring to these groups in R⁶, reference isalso made to any such groups that might be present in the R^(6a) andR^(6b) moieties.

Z is selected from a bond, —O—, —S—, —S(O)—, —SO₂—, —SO₂—NR^(Z1)—,—NR^(Z1)—SO₂—, —C(O)—, —OC(O)—, —C(O)O—, —NR^(Z1)C(O)—, —C(O)NR^(Z1)—,—NR^(Z2)—C(O)—NR^(Z3)—, —NR^(Z2)—C(S)—NR^(Z3)—, —CH(OH)—, and—C(═N—O—R^(Z4))—. In one embodiment, Z is selected from a bond, —O—, and—C(O)—. R^(Z1) is selected from H and —C₁₋₄alkyl. In one particularembodiment, R^(Z1) is hydrogen. R^(Z2) and R^(Z3) are independentlyselected from H, —C₁₋₄alkyl, and —C₃₋₆cycloalkyl. Alternately, R^(Z2)and R^(Z3) are taken together to form —C₂₋₄alkylene- or—C₂₋₃alkenylene-. In one particular embodiment, R^(Z2) and R^(Z3) areboth hydrogen. R^(Z4) is selected from —C₁₋₄alkyl and benzyl. In oneembodiment, R^(Z4) is —C₁₋₄alkyl such as —CH₃. In another embodiment,R^(Z4) is benzyl. Each of the aforementioned alkyl, alkylene,alkenylene, and cycloalkyl groups in Z may be substituted with 1 to 5fluoro atoms. It is understood that when referring to these groups in Z,reference is made to such groups that are present in the R^(Z1), R^(Z2),and R^(Z3) moieties.

The linker connecting the nitrogen-containing ring to the quaternarynitrogen may be a bond (0 carbon atoms) or may have 1 carbon atom, andthus may be designated as —(CH₂)₀₋₁— or —C₀₋₁alkylene-. In oneparticular embodiment, this linker is a bond. In another embodiment,this linker contains one carbon atom.

The linker connecting the quaternary nitrogen to the Z moiety containsfrom 1-4 carbon atoms, and thus may be designated as —(CH₂)₁₋₄— or—C₁₋₄alkylene-. Each —CH₂— group in —(CH₂)₁₋₄— is optionally substitutedwith 1 or 2 substituents independently selected from —C₁₋₂alkyl, —OH,fluoro, and phenyl. In one embodiment, one —CH₂— group in —(CH₂)₁₋₄— isoptionally substituted with —OH or phenyl.

These two linkers, in combination with the quaternary nitrogen and the Zmoiety, can be depicted as —(CH₂)₀₋₁—N(R⁵R⁶)—(CH₂)₁₋₄—Z—, and formingthe linking moiety between the nitrogen-containing ring and the Qmoiety. In specific embodiments, —(CH₂)₀₋₁—N(R⁵R⁶)—(CH₂)₁₋₄—Z— isselected from one of the following: —N(CH₃)₂—CH₂—, —N(CH₃)₂—(CH₂)₂—,—N(CH₃)₂—(CH₂)₃—, —N(CH₃)₂—(CH₂)₄—, —N(CH₃)₂—CH₂—CH(phenyl)-,—N(CH₃)₂—CH₂—CH(OH)—, —N(CH₃)₂—CH₂—C(O)—, —N(CH₃)₂—(CH₂)₂—O—,—N(CH₃)₂—(CH₂)₃—O—, —N(CH₃)₂—(CH₂)₄—O—, —CH₂—N(CH₃)₂—CH₂—,—CH₂—N(CH₃)₂—(CH₂)₂—, —CH₂—N(CH₃)₂—(CH₂)₃—, —N(CH₃)(CH₂CH₃)—(CH₂)₂—,—N(CH₃)—[(CH₂)₂CH₃]—(CH₂)₂—, —N(CH₃)(CH₂-cyclopropyl)-(CH₂)₂—,—N(CH₃)(CH₂CH₂OH)—(CH₂)₂—, —N(CH₃)[CH₂C(O)OCH₃]—(CH₂)₂—, and—N(CH₃)[CH₂C(O)NH₂]—(CH₂)₂—.

Q is an aryl or heteroaryl group, as those term are defined herein. Inone embodiment Q is an aryl group such as phenyl. In another embodiment,Q is a heteroaryl group selected from thienyl (e.g., thiophen-2-yl orthiophen-3-yl), furanyl, pyrrolyl (e.g., pyrrol-1-yl), pyrazolyl,benzo[1,3]dioxolyl, indolyl (e.g., 1H-indol-3-yl), and tetrazolyl (e.g.,1H-tetrazol-5-yl), and piperidinyl (e.g., piperidin-4-yl).

When Q is an aryl group, it may be substituted with 1 to 5 R^(Q) groupsindependently selected from halo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano,—C₀₋₂alkylene-COOH, —C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl, —S—C₁₋₄alkyl,—NH—C(O)—C₁₋₄alkyl, —N-di-C₁₋₄alkyl, and —N⁺(O)O. In another embodiment,each R^(Q) is independently selected from halo, —C₁₋₄alkyl,—C₀₋₄alkylene-OH, cyano, —C₀₋₂alkylene-COOH, —C(O)O—C₁₋₄alkyl, and—O—C₁₋₄alkyl. Exemplary halo groups include fluoro, chloro, and bromo.Exemplary —C₁₋₄alkyl groups include —CH₃ and —C(CH₃)₃, as well asfluoro-substituted alkyl groups such as —CF₃. Exemplary —C₀₋₄alkylene-OHgroups include —OH and —(CH₂)₂—OH. Exemplary —C₀₋₂alkylene-COOH groupsinclude —COOH (carboxy) and —CH₂COOH (carboxymethyl). Exemplary—C(O)O—C₁₋₄alkyl groups include —C(O)OCH₃ (methoxycarbonyl). Exemplary—O—C₁₋₄alkyl groups include —OCH₃, as well as fluoro-substituted alkoxygroups such as —OCHF₂ (difluoromethoxy). Exemplary —S—C₁₋₄alkyl groupsinclude —S—CH₃. Exemplary —NH—C(O)—C₁₋₄alkyl groups include—NH—C(O)—CH₃. Exemplary —N-di-C₁₋₄alkyl groups include —N(CH₃)₂.

When more than one R^(Q) substituent is present, the substituents can beon the same or on different ring atoms. Each of the aforementioned alkyland alkylene groups in Q may be substituted with 1 to 5 fluoro atoms. Itis understood that when referring to these groups in Q, reference ismade to such groups that are present in the R^(Q) moiety. In oneembodiment, Q is unsubstituted. In another embodiment, Q is substitutedwith one R^(Q) group; and in another embodiment, R^(Q) is selected fromhalo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano, —C₀₋₂alkylene-COOH,—C(O)O—C₁₋₄alkyl, and —O—C₁₋₄alkyl, and —N⁺(O)O. In another embodiment,Q is substituted with two R^(Q) groups; and in another embodiment, eachR^(Q) is independently selected from halo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH,—C(O)O—C₁₋₄alkyl, and —O—C₁₋₄alkyl.

X⁻ is an anion of a pharmaceutically acceptable acid. The term “anion ofa pharmaceutically acceptable acid” is used to refer to an anioniccounterion of a pharmaceutically acceptable acid. Examples ofpharmaceutically acceptable inorganic acids include, by way ofillustration and not limitation, boric, carbonic, hydrohalic(hydrobromic, hydrochloric, hydrofluoric or hydroiodic), nitric,phosphoric, sulfamic and sulfuric acids, and hydroxide. Examples ofpharmaceutically acceptable organic acids include, by way ofillustration and not limitation, aliphatic hydroxyl acids (e.g., citric,gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids),aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionicand trifluoroacetic acids), aromatic carboxylic acids (e.g., benzoic,p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylaceticacids), amino acids (e.g., aspartic and glutamic acids), aromatichydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic,1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylicacids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic andsuccinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic,pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,edisylic, ethanesulfonic, isethionic, methanesulfonic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,and the like. In one embodiment, the pharmaceutically acceptable acid isselected from acetic, benzenesulfonic, benzoic, butyric,p-chlorobenzoic, citric, diphenylacetic, formic, hydrobromic,hydrochloric, hydrofluoric, hydroiodic, o-hydroxybenzoic,p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic,3-hydroxynaphthalene-2-carboxylic, lactic, malic, maleic,methanesulfonic, nitric, phosphoric, propionic, succinic, sulfuric,tartaric, trifluoroacetic, and triphenylacetic acids. In anotherembodiment the pharmaceutically acceptable acid is selected fromhydrobromic, hydroiodic, and trifluoroacetic acids. In one embodiment,the anion is selected from acetate, benzenesulfonate, benzoate, bromide,butyrate, chloride, p-chlorobenzoate, citrate, diphenylacetate, formate,fluoride, o-hydroxybenzoate, p-hydroxybenzoate,1-hydroxynaphthalene-2-carboxylate, 3-hydroxynaphthalene-2-carboxylate,iodide, lactate, malate, maleate, methanesulfonate, nitrate, phosphate,propionate, succinate, sulfate, tartrate, trifluoroacetate, bi- andtriphenylacetate. In yet another embodiment, the anion is selected frombromide, iodide and trifluoroacetate.

In one embodiment the compounds of the invention have formula I or IIand: R¹ is selected from —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl,—C₁₋₃alkylene-SCH₃, —C₃₋₉cycloalkyl, and heteroaryl; R² is an aryl orheteroaryl group; R³ is selected from H and —C₀₋₁alkylene-OH; or when R¹is —C₃₋₉cycloalkyl, R³ can form a double bond with the carbon atom onthe —C₃₋₉cycloalkyl group; or —CR¹R² together form a group of formula:

a is 0; R⁵ is —C₁₋₅alkyl; R⁶ is selected from —C₁₋₃alkyl,—C₁₋₂alkyleneC₃₋₇cycloalkyl, —C₀₋₄alkylene-OH,—C₁₋₂alkylene-C(O)O—C₁₋₄alkyl, and —C₁₋₂alkylene-C(O)NH₂; Z is selectedfrom a bond, —O—, and —C(O)—; Q is an aryl or heteroaryl group; whereinthe aryl in R² is optionally substituted with 1 to 2 R groupsindependently selected from —C₁₋₄alkyl, halo, and —OR^(a); where eachR^(a) is independently selected from H and —C₁₋₄alkyl; the aryl in Q isoptionally substituted with 1 to 2 R^(Q) groups independently selectedfrom halo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano, —C₀₋₂alkylene-COOH,—C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl, and —N⁺(O)O; each alkyl in R^(Q) isoptionally substituted with 1 to 5 fluoro atoms; and one —CH₂— group in—(CH₂)₁₋₄— is optionally substituted with a group selected from —OH andphenyl.

In another embodiment the compounds of the invention have formula I orII and: R¹ is selected from —CH₂CH(CH₃)₂, —CH₂CH═CH₂, cyclopropyl, andcyclopentyl, when R² is phenyl and R³ is —OH; or R¹ is -cyclopropyl whenR² is phenyl and R³ is H; or R¹ is cyclopentyl when R² is phenyl and R³forms a double bond with the carbon atom on the cyclopentyl group; or R¹is cyclopentyl or thiophenyl, when R² is thiophenyl and R³ is —OH; or—CR¹R² together form a group of formula:

and: a is 0; the linker —(CH₂)₀₋₁—N(R⁵R⁶)—(CH₂)₁₋₄—Z— is selected from:—N(CH₃)₂—(CH₂)₂—, —N(CH₃)(CH₂CH₃)—(C₂)₂—, and —N(CH₃)₂—CH₂—C(O)—; Q isselected from phenyl, thiophen-2-yl, thiophen-3-yl, andbenzo[1,3]dioxol-5-yl; wherein the phenyl in R² is optionallysubstituted with 1 to 2 R groups independently selected from —CH₃,fluoro, —OH, and —OCH₃; the phenyl in Q is optionally substituted with 1to 2 R^(Q) groups independently selected from fluoro, bromo, —CH₃, and—OH.

In another embodiment of the invention, the quaternary ammonium compoundof formula II is the species embodied in formula IIa:

where R¹⁻⁶, a, Z, Q and X⁻ are as defined for formula I; andpharmaceutically acceptable salts thereof. In one particular embodiment,R¹ is selected from —CH₂CH(CH₃)₂, —CH₂CH═CH₂, —C≡CH, —(CH₂)₂SCH₃,cyclopropyl, cyclopentyl, and thiophenyl; R² is selected from phenyl andthiophenyl; R³ is selected from H and —C₀₋₁alkylene-OH; or when R¹ iscyclopentyl, R³ can form a double bond with the carbon atom on thecyclopentyl group; or —CR¹R² together form a group of formula:

a is 0; R⁵ is —CH₃; R⁶ is selected from —CH₃, —CH₂CH₃, —(CH₂)₂CH₃,—CH₂-cyclopropyl, —(CH₂)₂OH, —CH₂—C(O)OCH₃, and —CH₂—C(O)NH₂; Z isselected from a bond, —O—, and —C(O)—; Q is selected from phenyl,1H-indol-3-yl, thiophen-2-yl, thiophen-3-yl, benzo[1,3]dioxol-5-yl,pyrrol-1-yl, 1H-tetrazol-5-yl, and piperidin-4-yl; wherein the phenyl inR² is optionally substituted with 1 to 2 R groups independently selectedfrom —CH₃, fluoro, —OH, and —OCH₃; the phenyl in Q is optionallysubstituted with 1 to 2 R^(Q) groups independently selected from fluoro,chloro, bromo, —CH₃, —CF₃, —C(CH₃)₃, —OH, —(CH₂)₂—OH, cyano, —COOH,—CH₂COOH, C(O)OCH₃, —OCH₃, —OCHF₂, and —N⁺(O)O; and one —CH₂— group in—(CH₂)₁₋₄— is optionally substituted with 1 or 2 substituents selectedfrom —OH and phenyl.

In another embodiment of the invention, the quaternary ammonium compoundof formula II is the species embodied in formula IIb:

where R, R¹, R³, R⁵, R⁶, Z, Q and X⁻ are as defined for formula I; andpharmaceutically acceptable salts thereof. In one particular embodiment,R¹ is selected from —CH₂CH(CH₃)₂, —CH₂CH═CH₂, —C≡CH, —(CH₂)₂SCH₃,cyclopropyl, and cyclopentyl; R³ is selected from H —C₀₋₁alkylene-OH; orwhen R¹ is cyclopentyl, R³ can form a double bond with the carbon atomon the cyclopentyl group; R⁵ is —CH₃; R⁶ is selected from —CH₃, —CH₂CH₃,—(CH₂)₂CH₃, —CH₂-cyclopropyl, —(CH₂)₂OH, —CH₂—C(O)OCH₃, and—CH₂—C(O)NH₂; Z is selected from a bond, —O—, and —C(O)—; Q is selectedfrom phenyl, 1H-indol-3-yl, thiophen-2-yl, thiophen-3-yl,benzo[1,3]dioxol-5-yl, pyrrol-1-yl, 1H-tetrazol-5-yl, andpiperidin-4-yl; wherein R is independently selected from —CH₃, fluoro,—OH, and —OCH₃; the phenyl in Q is optionally substituted with 1 to 2R^(Q) groups independently selected from fluoro, chloro, bromo, —CH₃,—CF₃, —C(CH₃)₃, —OH, —(CH₂)₂—OH, cyano, —COOH, —CH₂COOH, C(O)OCH₃,—OCH₃, —OCHF₂, and —N⁺(O)O; and one —CH₂— group in —(CH₂)₁₋₄— isoptionally substituted with 1 substituent selected from —C₁₋₂alkyl, —OHand phenyl.

In another embodiment of the invention, the quaternary ammonium compoundof formula II is the species embodied in formula IIc:

where R, R¹, R³, R⁵, R⁶, Z, R^(Q) and X⁻ are as defined for formula I;and pharmaceutically acceptable salts thereof. In one particularembodiment, R¹ is selected from —CH₂CH(CH₃)₂, —CH₂CH═CH₂, —C≡CH,—(CH₂)₂SCH₃, cyclopropyl, and cyclopentyl; R³ is selected from H and—C₀₋₁alkylene-OH; or when R¹ is cyclopentyl, R³ can form a double bondwith the carbon atom on the cyclopentyl group; R⁵ is —CH₃; R⁶ isselected from —CH₃, —CH₂CH₃, —(CH₂)₂CH₃, —CH₂-cyclopropyl, —(CH₂)₂OH,—CH₂—C(O)OCH₃, and —CH₂—C(O)NH₂; Z is selected from a bond, —O—, and—C(O)—; wherein R is independently selected from —CH₃, fluoro, —OH, and—OCH₃; R^(Q) is independently selected from fluoro, chloro, bromo, —CH₃,—CF₃, —C(CH₃)₃, —OH, —(CH₂)₂—OH, cyano, —COOH, —CH₂COOH, C(O)OCH₃,—OCH₃, —OCHF₂, and —N⁺(O)O; and one —CH₂— group in —(CH₂)₁₋₄— isoptionally substituted with 1 substituent selected from —C₁₋₂alkyl, —OHand phenyl.

In another embodiment of the invention, the quaternary ammonium compoundof formula II is the species embodied in formula IId:

where R³, Z, R^(Q) and X⁻ are as defined for formula I; andpharmaceutically acceptable salts thereof. In one particular embodiment,R³ is —OH; Z is a bond; and R^(Q) is independently selected from fluoroand —OH.

In another embodiment of the invention, the quaternary ammonium compoundof formula II is the species embodied in formula IIe:

where R³, Z, Q and X⁻ are as defined for formula I; and pharmaceuticallyacceptable salts thereof. In one particular embodiment, R³ is —OH; Z isa bond; Q is phenyl or benzo[1,3]dioxol-5-yl; and the phenyl in Q isoptionally substituted with 1 R^(Q) group selected from fluoro and —CH₃.

In another embodiment of the invention, the quaternary ammonium compoundof formula II is the species embodied in formula IIf:

where Z, Q and X⁻ are as defined for formula I; and pharmaceuticallyacceptable salts thereof. In one particular embodiment, R³ is H; Z is abond; Q is phenyl, thiophen-2-yl, or thiophen-3-yl; and the phenyl in Qis optionally substituted with 1 R^(Q) group selected from fluoro and—OH.

A particular group of compounds of formula I are those disclosed in U.S.Provisional Application No. 60/925,951, filed on Apr. 24, 2007. Thisgroup includes compounds of formula (I′):

in salt or zwitterionic form, wherein: a′ is 0 or an integer of from 1to 5; each R¹ is independently selected from —C₁₋₄alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl, —C₃₋₆cycloalkyl, cyano, halo, —OR^(1a), —SR^(1a),—S(O)R^(1a), —S(O)₂R^(1a), and —NR^(1b)R^(1c); where each R^(1a) isindependently selected from H, —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl,and —C₃₋₆cycloalkyl; and each R^(1b) and R^(1c) is independentlyselected from H, —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, and—C₃₋₆cycloalkyl; Ar′ is an aryl group; R^(3′) is selected from H and—C₀₋₁alkylene-OH; or R³ forms a double bond with the carbon atom on the—C₅₋₉cycloalkyl group; b′ is 0 or an integer of from 1 to 3; each R^(4′)is independently selected from fluoro and —C₁₋₄alkyl; R^(5′) is selectedfrom —C₁₋₅alkyl and —C₀₋₁alkyleneC₃₋₅cycloalkyl; R^(6′) is selected from—C₁₋₃alkyl, —C₁₋₂alkyleneC₃₋₇cycloalkyl, —C₀₋₄alkylene-OH,—C₁₋₂alkylene-C(O)O—C₁₋₄alkyl, and —C₁₋₂alkylene-C(O)NR^(6a)R^(6b);where R^(6a) and R^(6b) are independently selected from H and—C₁₋₄alkyl; or R⁶ is taken with R⁵ to form —C₃₋₅alkylene-; Z′ isselected from a bond, —O—, —S—, —S(O)—, —SO₂—, —SO₂—NR^(Z1)—,—NR^(Z1)—SO₂—, —C(O)—, —OC(O)—, —C(O)O—, —NR^(Z1)C(O)—, —C(O)NR^(Z1)—,—NR^(Z2)—C(O)—NR^(Z3)—, —NR^(Z2)—C(S)—NR^(Z3)—, —CH(OH)—, and—C(═N—O—R^(Z4))—; where R^(Z1) is selected from H and —C₁₋₄alkyl; R^(Z2)and R^(Z3) are independently selected from H, —C₁₋₄alkyl, and—C₃₋₆cycloalkyl, or R^(Z2) and R^(Z3) are taken together to form—C₂₋₄alkylene- or —C₂₋₃alkenylene-; and R^(Z4) is selected from—C₁₋₄alkyl and benzyl; Q′ is an aryl or heteroaryl group; wherein Ar′ isoptionally substituted with 1 to 5 R^(2′) groups independently selectedfrom —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, —C₃₋₆cycloalkyl, cyano,halo, —OR^(2a), —SR^(2a), —S(O)R^(2a), —S(O)₂R^(2a), and —NR^(2b)R^(2c);where each R^(2a) is independently selected from H, —C₁₋₄alkyl,—C₂₋₄alkenyl, —C₂₋₄alkynyl, and —C₃₋₆cycloalkyl; and each R^(2b) andR^(2c) is independently selected from H, —C₁₋₄alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl, and —C₃₋₆cycloalkyl; wherein Q′ is optionally substitutedwith 1 to 5 R^(Q) groups independently selected from halo, —C₁₋₄alkyl,—C₀₋₄alkylene-OH, cyano, —C₀₋₂alkylene-COOH, —C(O)O—C₁₋₄alkyl,—O—C₁₋₄alkyl, —S—C₁₋₄alkyl, —NH—C(O)—C₁₋₄alkyl, —N-di-C₁₋₄alkyl, and—N⁺(O)O; wherein each alkyl, alkylene, alkenyl, alkenylene, alkynyl andcycloalkyl group in R^(1′-6′), Z′ and Q′ is optionally substituted with1 to 5 fluoro atoms; and each —CH₂— group in —(CH₂)₁₋₄— is optionallysubstituted with 1 or 2 substituents independently selected from—C₁₋₂alkyl, —OH, fluoro, and phenyl; and pharmaceutically acceptablesalts thereof.

In addition, particular compounds of formula I that are of interestinclude those set forth in the Examples below, as well as thepharmaceutically acceptable salts thereof.

Definitions

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to carbon atoms and include, for example—C₁₋₂alkyl, —C₁₋₃alkyl, —C₁₋₄alkyl, —C₁₋₅alkyl, and —C₁₋₆alkyl.Representative alkyl groups include, by way of example, methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl,n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown preceding the term assubscript. For example, the term “—C₁₋₄alkyl” means an alkyl grouphaving from 1 to 4 carbon atoms, where the carbon atoms are in anyacceptable configuration.

The term “alkylene” means a divalent saturated hydrocarbon group thatmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms and include, forexample, —C₀₋₁alkylene-, —C₀₋₂alkylene-, —C₀₋₄alkylene-, —C₀₋₅alkylene-,—C₁₋₂alkylene-, —C₁₋₄alkylene-, —C₂₋₄alkylene-, —C₂₋₅alkylene-,—C₃₋₅alkylene-, and —C₃₋₆alkylene-. Representative alkylene groupsinclude, by way of example, methylene, ethane-1,2-diyl (“ethylene”),propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyland the like. It is understood that when the alkylene term include zerocarbons such as —C₀₋₁alkylene- or —C₀₋₅alkylene-, such terms areintended to include a single bond.

The term “alkenyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon double bonds. Unless otherwisedefined, such alkenyl groups typically contain from 2 to carbon atomsand include, for example, —C₂₋₄alkenyl and —C₂₋₆alkenyl. Representativealkenyl groups include, by way of example, ethenyl, n-propenyl,isopropenyl, n-but-2-enyl, n-hex-3-enyl and the like. The term“alkenylene” means a divalent alkenyl group, and exemplary alkenylenegroups include —C₂₋₃alkenylene-.

The term “alkynyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon triple bonds. Unless otherwisedefined, such alkynyl groups typically contain from 2 to 10 carbon atomsand include, for example, —C₂₋₄alkynyl and —C₂₋₆alkynyl. Representativealkynyl groups include, by way of example, ethynyl, n-propynyl,n-but-2-ynyl, n-hex-3-ynyl and the like.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to,tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), and the like.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or fused rings (i.e., naphthalene). Unless otherwisedefined, such aryl groups typically contain from 6 to 10 carbon ringatoms and include, for example, —C₆₋₁₀aryl. Representative aryl groupsinclude, by way of example, phenyl and naphthalene-1-yl,naphthalene-2-yl, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms and include, for example,—C₃₋₅cycloalkyl, —C₃₋₆cycloalkyl, —C₃₋₇cycloalkyl, and —C₅₋₉cycloalkyl.Representative cycloalkyl groups include, by way of example,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term “divalent hydrocarbon group” means a divalent hydrocarbon groupwhich is composed primarily of carbon and hydrogen atoms and whichoptionally contains one or more heteroatoms. Such divalent hydrocarbongroups may be branched or unbranched, saturated or unsaturated, acyclicor cyclic, aliphatic or aromatic, or combinations thereof. The divalenthydrocarbon group can optionally contain heteroatoms incorporated intothe hydrocarbon chain or as substituents attached to the hydrocarbonchain.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heteroaryl” means a monovalent aromatic group having a singlering or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heteroaryl groups typicallycontain from 5 to 10 total ring atoms and include, for example,—C₂₋₉heteroaryl. Representative heteroaryl groups include, by way ofexample, monovalent species of pyrrole, imidazole, thiazole, oxazole,furan, thiophene, triazole, pyrazole, isoxazole, isothiazole, pyridine,pyrazine, pyridazine, pyrimidine, triazine, indole, benzofuran,benzothiophene, benzimidazole, benzthiazole, quinoline, isoquinoline,quinazoline, quinoxaline and the like, where the point of attachment isat any available carbon or nitrogen ring atom.

The term “leaving group” means a functional group or an atom that can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include, but are not limited to,chloro, bromo and iodo groups; sulfonic ester groups, such as mesylate,tosylate, brosylate, nosylate and the like; and acyloxy groups, such asacetoxy, trifluoroacetoxy and the like.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise undesirable. For example, the term“pharmaceutically acceptable carrier” refers to a material that can beincorporated into a composition and administered to a patient withoutcausing undesirable biological effects or interacting in a deleteriousmanner with other components of the composition. Such pharmaceuticallyacceptable materials typically have met the required standards oftoxicological and manufacturing testing, and include those materialsidentified as suitable inactive ingredients by the U.S. Food and Drugadministration.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, e.g., a compound of formula I or apharmaceutically acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include, by way of example, water, methanol, ethanol,isopropanol, acetic acid and the like. When the solvent is water, thesolvate formed is a hydrate.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.In particular, an “effective” amount is that amount needed to obtain thedesired result, and a “therapeutically effective” amount is that amountneeded to obtain the desired therapeutic effect. For example, forantagonizing a muscarinic receptor, an “effective amount” is amuscarinic receptor-antagonizing amount. Similarly, a therapeuticallyeffective amount for treating chronic obstructive pulmonary disease(COPD) is that amount that will achieve the desired therapeutic result,which may be disease prevention, amelioration, suppression oralleviation.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD or asthma) ina patient, such as a mammal (particularly a human) that includes: (a)preventing the disease or medical condition from occurring, i.e.,prophylactic treatment of a patient; (b) ameliorating the disease ormedical condition, i.e., eliminating or causing regression of thedisease or medical condition in a patient; (c) suppressing the diseaseor medical condition, i.e., slowing or arresting the development of thedisease or medical condition in a patient; or (d) alleviating thesymptoms of the disease or medical condition in a patient. For example,the term “treating COPD” would include preventing COPD from occurring,ameliorating COPD, suppressing COPD, and alleviating the symptoms ofCOPD. The term “patient” is intended to include those animals, such ashumans, that are in need of treatment or disease prevention, that arepresently being treated for disease prevention or treatment of aspecific disease or medical condition, as well as test subjects in whichcompounds of the invention are being evaluated or being used in a assay,for example an animal model.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods, the proceduresset forth in the Examples, or by using other methods, reagents, andstarting materials that are known to those of ordinary skill in the art.Although the following procedures may illustrate a particular embodimentof the invention, it is understood that other embodiments of theinvention can be similarly prepared using the same or similar methods orby using other methods, reagents and starting materials known to thoseof ordinary skill in the art. It will also be appreciated that wheretypical or preferred process conditions (i.e., reaction temperatures,times, mole ratios of reactants, solvents, pressures, etc.) are given,other process conditions can also be used unless otherwise stated. Whileoptimum reaction conditions will typically vary depending on variousreaction parameters such as the particular reactants, solvents andquantities used, those of ordinary skill in the art can readilydetermine suitable reaction conditions using routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well-known in the art.Functional groups that may be protected so as to prevent undesiredreactions include, by way of example, carboxy groups, amino groups,hydroxyl groups, thiol groups, carbonyl groups and the like.Representative carboxy-protecting groups include, but are not limitedto, esters, such as methyl, ethyl, t-butyl, benzyl (Bn), p-methoxybenzyl(PMB), 9-fluoroenylmethyl (Fm), trimethylsilyl (TMS),t-butyldimethylsilyl (TBS), diphenylmethyl (benzhydryl, DPM) and thelike; amides and hydrazides. Representative protecting groups for aminogroups include carbamates (such as tert-butoxycarbonyl) and amides.Representative hydroxyl-protecting groups include, but are not limitedto, silyl groups including triC₁₋₆alkylsilyl groups, such astrimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBS)and the like; esters (acyl groups) including C₁₋₆alkanoyl groups, suchas formyl, acetyl and the like; arylmethyl groups, such as benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), diphenylmethyl(benzhydryl, DPM) and the like; and ethers. Representative protectinggroups for thiol groups include thioethers and thioesters.Representative protecting groups for carbonyl groups include acetals andketals. Protecting groups other than those described herein may be used,if desired. For example, numerous protecting groups, and theirintroduction and removal, are described in T. W. Greene and G. M. Wuts,Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York,1999, and references cited therein.

By way of illustration, compounds of formula I can be prepared by one ormore of the following exemplary processes: (a) reacting a compound offormula 1 with a compound of formula 2:

or reacting a compound of formula 1′ with a compound of formula 2′:

where L¹ represents a leaving group, to produce a compound of formula 3:

and reacting the compound of formula 3 with an organic substratecontaining an R⁶ group; or (b) reacting a compound of formula 4:

with a compound of formula 2; or (c) reacting a compound of formula 4with a compound of formula 5:

where L² represents a leaving group and A is defined below, to produce acompound of formula 6:

and reacting the compound of formula 6 with a compound of formula 7:B-Q  (7)where Z, A and B are defined as set forth in the following table, and L³represents a leaving group:

Z A B bond —CH₃ L³— —O— —L³ HO— —S— —L³ HS— —SO₂—NR^(Z1)— —SO₂—OH or—SO₂Cl R^(Z1)HN— —NR^(Z1)—SO₂— —NHR^(Z1) HOO₂S— —OC(O)— —OH HO(O)C——C(O)O— —C(O)OH or —C(O)Cl HO— —NR^(Z1)C(O)— —NHR^(Z1) HO(O)C—or Cl(O)C——NR^(Z2)—C(O)—NR^(Z3)—, —N═C═O H₂N— where R^(Z2) and R^(Z3) are HNR^(Z2)—C(S)—NR^(Z3)—, —N═S═O H₂N— where R^(Z2) and R^(Z2) are Hand recovering the product in salt or zwitterionic form.

The resulting reaction product, a compound of formula I, is a quaternaryammonium compound. This compound can be crystallized by first convertingthe compound to the appropriate counterion form, then crystallizing thecompound in a suitable solvent. Such crystals are quaternary ammoniumsalts.

In these reactions, depending upon the particular substituents present,one or more protecting groups may be employed. If such protecting groupsare used, they are removed using conventional procedures to provide thecompound of formula I.

Process (a)

In process (a), the reaction between the compounds (1) and (2), theleaving group represented by L¹ can be, for example, halo, such aschloro, bromo or iodo, or a sulfonic ester group, such as mesylate ortosylate. In one embodiment, L¹ is bromo. The reaction is convenientlyperformed in the presence of a base, for example, a tertiary amine suchas diisopropylethylamine. Convenient solvents include nitriles, such asacetonitrile, dimethylformamide (DMF), and dimethylacetamide (DMA). Thereaction is conveniently conducted at a temperature in the range of from0° C. to 100° C. The reaction product is then isolated usingconventional procedures, such as extraction, recrystallization,chromatography and the like.

Compound (3), the free base form of the desired product, is dissolved ina suitable solvent then contacted with an organic substrate. Exemplarysolvents include toluene, DMA, and CH₃CN. The organic substrate istypically a pharmaceutically acceptable acid such as an organic halide.The substrate contains an R⁶ group, for example, —C₁₋₆alkyl which may besubstituted with 1-5 fluoro atoms, and a leaving group, examples ofwhich include halides such as iodide or bromide. Exemplary substratesinclude methyl iodide, methyl bromide, ethyl iodide, propyl iodide,benzyl bromide and benzyl iodide.

In some situations, process (a) can be followed by a second reaction toyield a different compound of formula I. For example, compounds where Zis —S(O)— or —SO₂— can be made by forming a compound of formula I whereZ is —S—, and subjecting such compound to an oxidation reaction. Inaddition, compounds where Z is —C(═N—O—R^(Z4))— can be made by forming acompound of formula I where Z is —C(O)—, and subjecting such compound toan imine formation reaction with H₂N—O—R^(Z4).

Compound (1) is generally known in the art or can be prepared fromcommercially available starting materials and reagents using well-knownprocedures. For example, compound (1) may be prepared by the followingreaction:

in which P¹ represents an amino-protecting group, such as a benzylgroup. Benzyl groups are conveniently removed by reduction, for example,using a hydrogen or ammonium formate and a Group VIII metal catalyst,such as palladium. Optionally, this reaction is conducted in thepresence of an acid, such as formic acid, acetic acid, hydrochloricacid, hydrobromic acid, sulfuric acid and the like. Examples of compound(1′) include (R)-cyclopentylhydroxyphenyl acetic acid,9H-Xanthene-9-carboxylic acid, and α-cyclopentylphenyl acetic acid.These compounds are commercially available or can be readily prepared,and exemplary preparation techniques are described in the examplessection.

Compound (2) is generally known and commercially available, or can beprepared from readily available starting materials using well-knownsynthetic methods. Examples of compound (2) include1-bromo-2-phenylethane, 3-(2-bromoethyl)phenol, 4-(2-bromoethyl)phenol,and 1-(2-bromoethyl)-2-fluorobenzene.

Alternately, compound (3) can be produced by coupling compounds (1′) and(2′) under conventional amide bond-forming conditions. Compound (2′) isgenerally known in the art or can be prepared from commerciallyavailable starting materials and reagents using well-known procedures.For example, compound (2′) may be prepared by the following reaction:

in which P¹ represents an amino-protecting group L¹ represents a leavinggroup.

Process (b)

In process (b), the reaction between the compounds (4) and (2) can beconducted using known procedures for reacting pyrrolidines withhalogenated compounds. The reaction is typically conducted in an organicsolvent at a temperature in the range of from about 20 to 120° C., moretypically in the range of about from about 20 to 80° C. Suitable organicsolvents include acetonitrile, dimethylsulfoxide, N,N-dimethylformamide(DMF), N,N-dimethylacetamide (DMA), ether, and acetone.

Examples of compound (4) include:(R)-2-cyclopentyl-1-(4-dimethylamino-piperidin-1-yl)-2-hydroxy-2-phenylethanone;2-cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-thiophen-2-ylethanone;1-(4-dimethylamino-piperidin-1-yl)-2-hydroxy-4-methyl-2-phenylpentan-1-one;(4-dimethylaminopiperidin-1-yl)(9H-xanthen-9-yl)-methanone;1-(4-dimethylamino-piperidin-1-yl)-2-hydroxy-2,2-di-thiophen-2-ylethanone;2-cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-phenylethanone; and2-cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-p-tolylethanone.

Compound (4) may be formed by a coupling reaction of compound (1′) and(4a) under conventional amide bond-forming conditions:

Suitable carboxylic acid/amine coupling reagents includebenzotriazol-1-yloxytris-(dimethylamino)phosphonium hexafluorophosphate(BOP), benzotriazol-1-yloxy-tripyrrolidinophosphoniumhexafluorophosphate (PyBOP),O-(7-azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), 1-hydroxybenzotriazole hydrate (HOBt),dicyclohexylcarbodiimide (DCC),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI),carbonyldiimidazole (CDI), and the like. Coupling reactions areconducted in an inert solvent such as DCM in the presence of one or morecoupling reagents and a base such as DIPEA, and are performed underconventional amide bond-forming conditions. Note that a coupling reagentmay not be required. For example, compounds (1′) and (4a) can be coupledusing DMF as the solvent and DIPEA as the base.

Alternately, compound (4), where R³ is —OH, may be formed by a couplingreaction of compound (4a) and (4b), under conventional amidebond-forming conditions, followed by reaction with R³—MgBr:

Examples of compound (4b) include 2-thiopheneglyoxylic acid,benzoylformic acid, and 4-methylphenyl)(oxo)acetic acid. Examples ofR³—MgBr include cyclopentyl magnesium bromide, thiophen-2-yl magnesiumbromide and isobutyl magnesium bromide.

Process (c)

In process (c), the reaction conditions between compound (4), (5) and(6) will vary depending upon the respective A and B groups. The leavinggroup represented by L³ can be, for example, a halo, typically bromo.Compound (5) and (6) are generally known or can be prepared from readilyavailable starting materials using well-known synthetic methods.

Some reactions in process (c) are coupling reactions, for example, whenZ is —NR^(Z1)C(O)—. In those reactions, the acidic moiety-containingcompound may be in the form of a reactive derivative. For example, thecarboxylic acid may be activated, for example, by forming an anhydrideor carboxylic acid halide, such as a carboxylic acid chloride. Thus thecarboxylic acid chloride is a reactive derivative of carboxylic acid.Alternatively, the carboxylic acid can be activated using conventionalcarboxylic acid/amine coupling reagents, such carbodiimides,O-(7-azabenzotriazol-1-yl-N,N,N′,N′ tetramethyluroniumhexafluorophosphate (HATU) and the like. The sulfonic acid and thio acidmoieties can be similarly derivatized. The reactions are conducted underconventional conditions using suitable coupling agents such ascarbonyldiimidazole. The reaction is typically conducted in the presenceof solvents such as trifluoroacetic acid and dichloromethane, andconveniently conducted at a temperature in the range of from −10° C. to100° C.

The remaining reactions in process (c) are alkylation reactions, forexample, when Z is —O—. The reactions are conducted under conventionalconditions using suitable solvents such as DMF or DMA, and convenientlyconducted at a temperature in the range of from room temperature to 100°C. In addition, process (c) illustrates formation compounds of formula Iwhere the R^(Z2) and/or R^(Z3) moieties are hydrogen. Such compounds arereadily converted to compound (1) where R^(Z2) and/or R^(Z3) are—C₁₋₄alkyl or —C₃₋₆cycloalkyl, or are taken together to form an—C₂₋₄alkylene- or —C₂₋₃alkenylene-linkage.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth below.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to,inhaled, oral, nasal, topical (including transdermal) and parenteralmodes of administration. Further, the compounds of the invention may beadministered, for example orally, in multiple doses per day, in a singledaily dose or a single weekly dose. It will be understood that any formof the compounds of the invention, (i.e., free base, pharmaceuticallyacceptable salt, solvate, etc.) that is suitable for the particular modeof administration can be used in the pharmaceutical compositionsdiscussed herein.

Accordingly, in one embodiment, the invention is directed to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the invention. The compositions may containother therapeutic and/or formulating agents if desired. A “compound ofthe invention” may also be referred to herein as the “active agent.”

The pharmaceutical compositions of this invention typically contain atherapeutically effective amount of a compound of the invention. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,i.e., bulk compositions, or less than a therapeutically effectiveamount, i.e., individual unit doses designed for multiple administrationto achieve a therapeutically effective amount. In one embodiment, thecomposition will contain from about 0.01-95 wt % of active agent,including, from about 0.01-30 wt %, such as from about 0.01-10 wt %,with the actual amount depending upon the formulation itself, the routeof administration, the frequency of dosing, and so forth. In anotherembodiment, a composition suitable for inhalation, for example,comprises from about 0.01-30 wt % or active agent with yet anotherembodiment comprises from about 0.01-10 wt % active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers and the like usingconventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable forinhaled administration. Suitable compositions for inhaled administrationwill typically be in the form of an aerosol or a powder. Suchcompositions are generally administered using well-known deliverydevices, such as a nebulizer inhaler, a dry powder inhaler, or ametered-dose inhaler, examples of which are described below.

In a specific embodiment of the invention, a composition comprising theactive agent is administered by inhalation using a nebulizer inhaler.Such nebulizer devices typically produce a stream of high velocity airthat causes the composition to spray as a mist that is carried into apatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the active agent is typically dissolved in a suitablecarrier to form a solution. Alternatively, the active agent can bemicronized and combined with a suitable carrier to form a suspension ofmicronized particles of respirable size, where micronized is typicallydefined as having particles in which at least about 90 percent of theparticles have a mass median diameter of less than about 10 μm. The term“mass median diameter” means the diameter such that half the mass of theparticles is contained in particles with larger diameter and half iscontained in particles with smaller diameter.

Suitable nebulizer devices include the Respimat® Soft Mist™ Inhaler(Boehringer Ingelheim), the AERx® Pulmonary Delivery System (AradigmCorp.), and the PARI LC Plus Reusable Nebulizer (Pari GmbH). Anexemplary composition for use in a nebulizer inhaler comprises anisotonic aqueous solution comprising from about 0.05 μg/mL to about 10mg/mL of a compound of the invention. In one embodiment, such a solutionhas a pH of about 4-6.

In another specific embodiment of the invention, a compositioncomprising the active agent is administered by inhalation using a drypowder inhaler (DPI). Such DPIs typically administer the active agent asa free-flowing powder that is dispersed in a patient's air-stream duringinspiration. In order to achieve a free flowing powder, the active agentis typically formulated with a suitable excipient such as lactose,starch, mannitol, dextrose, polylactic acid, polylactide-co-glycolide,and combinations thereof. Typically, the active agent is micronized andcombined with an excipient to form a blend suitable for inhalation.Accordingly, in one embodiment of the invention, the active agent is inmicronized form. For example, a representative composition for use in aDPI comprises dry lactose having a particle size between about 1 μm andabout 100 μm (e.g., dry milled lactose) and micronized particles of theactive agent. Such a dry powder formulation can be made, for example, bycombining lactose with the active agent and then dry blending thecomponents. Alternatively, if desired, the active agent can beformulated without an excipient. The composition is then typicallyloaded into a DPI, or into inhalation cartridges or capsules for usewith a DPI. DPIs are well known to those of ordinary skill in the art,and many such devices are commercially available, with representativedevices including Aerolizer® (Novartis), Airmax™ (IVAX), ClickHaler®(Innovata Biomed), Diskhaler® (GlaxoSmithKline), Diskus® or Accuhaler(GlaxoSmithKline), Easyhaler® (Orion Pharma), Eclipse™ (Aventis),FlowCaps® (Hovione), Handihaler® (Boehringer Ingelheim), Pulvinal®(Chiesi), Rotahaler® (GlaxoSmithKline), SkyeHaler™ or Certihaler™(SkyePharma), Twisthaler (Schering-Plough), Turbuhaler® (AstraZeneca),Ultrahaler® (Aventis), and the like.

In yet another specific embodiment of the invention, the compositioncomprising the active agent is administered by inhalation using ametered-dose inhaler (MDI). Such MDIs typically discharge a measuredamount of the active agent using compressed propellant gas. Metered-doseformulations thus typically comprise a solution or suspension of theactive agent in a liquefied propellant, such as a chlorofluorocarbonsuch as CCl₃F or a hydrofluoroalkane (HFA) such as1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane (HFA 227), although HFAs aregenerally preferred due to concerns about chlorofluorocarbons affectingthe ozone layer. Additional optional components of HFA formulationsinclude co-solvents, such as ethanol or pentane, and surfactants, suchas sorbitan trioleate, oleic acid, lecithin, and glycerin. See, forexample, U.S. Pat. No. 5,225,183 to Purewal et al., EP 0717987 A2(Minnesota Mining and Manufacturing Company), and WO 92/22286 (MinnesotaMining and Manufacturing Company). A representative composition for usein an MDI comprises from about 0.01-5 wt % of active agent; from about0-20 wt % ethanol; and from about 0-5 wt % surfactant; with theremainder being an HFA propellant. Such compositions are typicallyprepared by adding a chilled or pressurized hydrofluoroalkane to asuitable container containing the active agent, ethanol (if present) andthe surfactant (if present). To prepare a suspension, the active agentis micronized and then combined with the propellant. The formulation isthen loaded into an aerosol canister, which forms a portion of the MDI.MDIs are well known to those of ordinary skill in the art, and many suchdevices are commercially available, with representative devicesincluding AeroBid Inhaler System (Forest Pharmaceuticals), AtroventInhalation Aerosol (Boehringer Ingelheim), Flovent® (GlaxoSmithKline),Maxair Inhaler (3M), Proventil® Inhaler (Schering), Serevent® InhalationAerosol (GlaxoSmithKline), and the like. Alternatively, a suspensionformulation can be prepared by spray drying a coating of surfactant onmicronized particles of the active agent. See, for example, WO 99/53901(Glaxo Group Ltd.) and WO 00/61108 (Glaxo Group Ltd.). Additionalexamples of processes of preparing respirable particles, andformulations and devices suitable for inhalation dosing are described inU.S. Pat. No. 5,874,063 to Briggner et al.; U.S. Pat. No. 5,983,956 toTrofast; U.S. Pat. No. 6,221,398 to Jakupovic et al.; U.S. Pat. No.6,268,533 to Gao et al.; U.S. Pat. No. 6,475,524 to Bisrat et al.; andU.S. Pat. No. 6,613,307 to Cooper.

In another embodiment, the pharmaceutical compositions are suitable fororal administration. Suitable compositions for oral administration maybe in the form of capsules, tablets, pills, lozenges, cachets, dragees,powders, granules; solutions or suspensions in an aqueous or non-aqueousliquid; oil-in-water or water-in-oil liquid emulsions; elixirs orsyrups; and the like; each containing a predetermined amount of theactive agent.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the composition will typicallycomprise the active agent and one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate. Solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; solution retarding agents, such as paraffin;absorption accelerators, such as quaternary ammonium compounds; wettingagents, such as cetyl alcohol and/or glycerol monostearate; absorbents,such as kaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, if appropriate, with oneor more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Suspensions maycontain suspending agents such as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, i.e., each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

Compounds of the invention can also be administered parenterally (e.g.,by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agent is provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. A typicalparenteral formulation is a sterile pH 4-7 aqueous solution of theactive agent. Parenteral formulations may also contain one or moresolubilizers, stabilizers, preservatives, wetting agents, emulsifiers,and dispersing agents. These formulations may be rendered sterile by useof a sterile injectable medium, a sterilizing agent, filtration,irradiation, or heat.

Compounds of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, thecompound can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

If desired, the compounds of this invention may be administered incombination with one or more other therapeutic agents. Thus, in oneembodiment, compositions of the invention may optionally contain otherdrugs that are co-administered with a compound of the invention. Forexample, the composition may further comprise one or more drugs (alsoreferred to as “secondary agents(s)”) selected from the group of otherbronchodilators (e.g., PDE₃ inhibitors, adenosine 2b modulators and β₂adrenergic receptor agonists); anti-inflammatory agents (e.g., steroidalanti-inflammatory agents such as corticosteroids and glucocorticoids;non-steroidal anti-inflammatory agents (NSAIDs); and PDE₄ inhibitors);other muscarinic receptor antagonists (i.e., antichlolinergic agents);antiinfective agents (e.g., Gram positive and Gram negative antibiotics,and antiviral agents); antihistamines; protease inhibitors; afferentblockers (e.g., D₂ agonists and neurokinin modulators); and combinationsthereof. Numerous examples of such therapeutic agents are well known inthe art, and examples are described below. By combining a compound ofthe invention with a secondary agent, double therapy can be achieved,i.e., muscarinic receptor antagonist activity and activity associatedwith the secondary agent (e.g., β₁ adrenergic receptor agonist), in somecases by administering two compositions and in some cases byadministering a single composition containing the active agent and thesecondary agent. Accordingly, in yet another aspect of the invention, apharmaceutical composition comprises a compound of the invention, asecond active agent, and a pharmaceutically acceptable carrier. Third,fourth etc. active agents may also be included in the composition. Forexample, a composition may comprise a compound of the invention; asecondary agent selected from corticosteroids, β₂ adrenergic receptoragonists; phosphodiesterase-4 inhibitors, and combinations thereof; anda pharmaceutically acceptable carrier. In a specific embodiment, thecomposition comprises a compound of the invention, a β₂ adrenergicreceptor agonist, and a steroidal anti-inflammatory agent. Incombination therapy, the amount of compound of the invention that isadministered, as well as the amount of secondary agents, may be lessthan the amount typically administered in monotherapy.

A compound of the invention may be either physically mixed with thesecond active agent to form a composition containing both agents; oreach agent may be present in separate and distinct compositions whichare administered to the patient simultaneously or sequentially. Forexample, a compound of the invention can be combined with a secondactive agent using conventional procedures and equipment to form acombination of active agents comprising a compound of the invention anda second active agent. Additionally, the active agents may be combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition comprising a compound of the invention, a second activeagent and a pharmaceutically acceptable carrier. In this embodiment, thecomponents of the composition are typically mixed or blended to create aphysical mixture. The physical mixture is then administered in atherapeutically effective amount using any of the routes describedherein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention. In other embodiments this timed relationshipis less than 12 hours, less than 8 hours, less than 6 hours, less than 4hours, less than 3 hours, less than 1 hour, less than thirty minutes,less than ten minutes, less than one minute, or immediately afteradministration of the compound of the invention. This is also referredto as sequential administration. Thus, a compound of the invention canbe administered by inhalation simultaneously or sequentially withanother active agent using an inhalation delivery device that employsseparate compartments (e.g. blister packs) for each active agent, wheresequential may mean being administered immediately after administrationof the compound of the invention or at some predetermined time later(e.g., one hour later or three hours later). Alternatively, thecombination may be administered using separate delivery devices, i.e.,one delivery device for each agent. Additionally, the agents can bedelivered by different routes of administration, i.e., one by inhalationand the other by oral administration.

In one embodiment, the kit comprises a first dosage form comprising acompound of the invention and at least one additional dosage formcomprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc,) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount. i.e., are typically administered in an amount thatproduces a therapeutically beneficial effect when co-administered with acompound of the invention. The secondary agent can be in the form of apharmaceutically acceptable salt, solvate, optically pure stereoisomer,and so forth. Thus, secondary agents listed below are intended toinclude all such forms, and are commercially available or can beprepared using conventional procedures and reagents. Suitable doses fora secondary agent are typically in the range of about 0.05 μg/day toabout 500 mg/day.

In a particular embodiment, a compound of the invention is administeredin combination with a β₂ adrenergic receptor agonist. Representative β₂adrenergic receptor agonists include, but are not limited to, albuterol,bitolterol, fenoterol, formoterol, indacaterol, isoetharine,levalbuterol, metaproterenol, pirbuterol, salbutamol, salmefamol,salmeterol, terbutaline, and the like. Other β₂ adrenergic receptoragonists that can be used in combination with compounds of the inventioninclude, but are not limited to,3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)-benzenesulfonamideand3-(-3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}amino)heptyl]oxy}propyl)benzenesulfonamideand related compounds disclosed in WO 02/066422 (Glaxo Group Ltd.);3-[3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)-phenyl]imidazolidine-2,4-dioneand related compounds disclosed in WO 02/070490 (Glaxo Group Ltd.);3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}-butyl)benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamide,N-(t-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,N-(t-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamideand related compounds disclosed in WO 02/076933 (Glaxo Group Ltd.);4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)-oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenoland related compounds disclosed in WO 03/024439 (Glaxo Group Ltd.);N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)-ethylamineand related compounds disclosed in U.S. Pat. No. 6,576,793 to Moran etal.;N-{2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-(R)-2-hydroxy-2-(8-hydroxy-2(1H)-quinolinon-5-yl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,653,323 to Moran etal. In a particular embodiment, the β₂-adrenoreceptor agonist is acrystalline monohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)-phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamine.Typically, the β₂-adrenoreceptor agonist will be administered in anamount sufficient to provide from about 0.05-500 μg per dose.

In a particular embodiment, a compound of the invention is administeredin combination with a steroidal anti-inflammatory agent. Representativesteroidal anti-inflammatory agents include, but are not limited to,beclomethasone dipropionate; budesonide; butixocort propionate;20R-16α,17α-[butylidenebis(oxy)]-6α,9α-difluoro-11β-hydroxy-17β-(methylthio)androsta-4-en-3-one(RPR-106541); ciclesonide; dexamethasone;6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester;6α,9α-difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester;6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxyandrosta-1,4-diene-17β-carbothioicacid (S)-(2-oxotetra-hydrofuran-3S-yl)ester; flunisolide; fluticasonepropionate; methyl prednisolone; mometasone furoate; prednisolone;prednisone; rofleponide; ST-126; triamcinolone acetonide; and the like.Typically, the steroidal anti-inflammatory agent will be administered inan amount sufficient to provide from about 0.05-500 μg per dose.

An exemplary combination is a compound of the invention co-administeredwith salmeterol as the β2 adrenergic receptor agonist, and fluticasonepropionate as the steroidal anti-inflammatory agent. Another exemplarycombination is a compound of the invention co-administered with acrystalline monohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)-ethylamineas the β₂-adrenoreceptor agonist, and6α,9α-difluoro-17α-[(2-furanylcarbonyl)-oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester as the steroidal anti-inflammatory agent.

Other suitable combinations include, for example, otheranti-inflammatory agents, e.g., NSAIDs (such as sodium cromoglycate;nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g.,theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors);leukotriene antagonists (e.g., monteleukast); inhibitors of leukotrienesynthesis; iNOS inhibitors; protease inhibitors, such as tryptase andelastase inhibitors; beta-2 integrin antagonists and adenosine receptoragonists or antagonists (e.g., adenosine 2a agonists); cytokineantagonists (e.g., chemokine antagonists such as, an interleukinantibody (αIL antibody), specifically, an αIL-4 therapy, an αIL-13therapy, or a combination thereof); or inhibitors of cytokine synthesis.

In a particular embodiment, a compound of the invention is administeredin combination with a phosphodiesterase-4 (PDE4) inhibitors or mixedPDE3/PDE4 inhibitors. Representative PDE4 or mixed PDE3/PDE4 inhibitorsinclude, but are not limited to, c is4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)-cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)-cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds disclosed inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds disclosed in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline(Almirall-Prodesfarma); VM554/UM565 (Vernalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

In a particular embodiment, a compound of the invention is administeredin combination with a muscarinic antagonist (i.e., anticholinergicagent). Representative muscarinic antagonists include, but are notlimited to, atropine, atropine sulfate, atropine oxide, methylatropinenitrate, homatropine hydrobromide, hyoscyamine (d, l) hydrobromide,scopolamine hydrobromide, ipratropium bromide, oxitropium bromide,tiotropium bromide, methantheline, propantheline bromide, anisotropinemethyl bromide, clidinium bromide, copyrrolate (Robinul), isopropamideiodide, mepenzolate bromide, tridihexethyl chloride (Pathilone),hexocyclium methylsulfate, cyclopentolate hydrochloride, tropicamide,trihexyphenidyl hydrochloride, pirenzepine, telenzepine, AF-DX 116 andmethoctramine and the like.

In a particular embodiment, a compound of the invention is administeredin combination with an antihistamine (i.e., H₁-receptor antagonist).Representative antihistamines include, but are not limited to,ethanolamines, such as carbinoxamine maleate, clemastine fumarate,diphenylhydramine hydrochloride and dimenhydrinate; ethylenediamines,such as pyrilamine amleate, tripelennamine hydrochloride andtripelennamine citrate; alkylamines, such as chlorpheniramine andacrivastine; piperazines, such as hydroxyzine hydrochloride, hydroxyzinepamoate, cyclizine hydrochloride, cyclizine lactate, meclizinehydrochloride and cetirizine hydrochloride; piperidines, such asastemizole, levocabastine hydrochloride, loratadine or itsdescarboethoxy analogue, terfenadine and fexofenadine hydrochloride;azelastine hydrochloride; and the like.

The following formulations illustrate representative pharmaceuticalcompositions of the invention.

Exemplary Compositions for Administration by a DPI

A compound of the invention (0.2 mg) is micronized and then blended withlactose (25 mg). This blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a DPI, for example.

A micronized compound of the invention (100 mg) is blended with milledlactose (25 g) (e.g., lactose in which not greater than about 85% of theparticles have a MMD of about 60 μm to about 90 μm and not less than 15%of the particles have a MMD of less then 15 μm). This blended mixture isthen loaded into individual blisters of a peelable blister pack in anamount sufficient to provide about 10 μg to about 500 μg of the compoundof the invention per dose. The contents of the blisters are administeredusing a DPI.

A micronized compound of the invention (1 g) is blended with milledlactose (200 g) to form a bulk composition having a weight ratio ofcompound to milled lactose of 1:200. The blended composition is packedinto a DPI capable of delivering between about 10 μg to about 500 μg ofthe compound of the invention per dose.

A micronized compound of the invention (100 mg) and a micronized β₂adrenergic receptor agonist (500 mg) are blended with milled lactose (30g). The blended mixture is then loaded into individual blisters of apeelable blister pack in an amount sufficient to provide about 10 μg toabout 500 μg of the compound of the invention per dose. The contents ofthe blisters are administered using a DPI.

Exemplary Compositions for Use in an MDI

A micronized compound of the invention (10 g) is dispersed in a solutionprepared by dissolving lecithin (0.2 g) in demineralized water (200 mL).The resulting suspension is spray dried and then micronized to form amicronized composition comprising particles having a mean diameter lessthan about 1.5 μm. The micronized composition is then loaded into MDIcartridges containing pressurized 1,1,1,2-tetrafluoroethane in an amountsufficient to provide about 10 μg to about 500 μg of the compound of theinvention per dose when administered by the MDI.

A suspension containing 5 wt % compound of the invention, 0.5 wt %lecithin, and 0.5 wt % trehalose is prepared by dispersing 5 g of acompound of the invention as micronized particles with mean size lessthan 10 μm in a colloidal solution formed from 0.5 g of trehalose and0.5 g of lecithin dissolved in 100 mL of demineralized water. Thesuspension is spray dried and the resulting material is micronized toparticles having a mean diameter less than 1.5 μm. The particles areloaded into canisters with pressurized 1,1,1,2-tetrafluoroethane.

Exemplary Composition for Use in a Nebulizer Inhaler

A compound of the invention (25 mg) is dissolved in citrate buffered (pH5) isotonic saline (125 mL). The mixture is stirred and sonicated untilthe compound is dissolved. The pH of the solution is checked andadjusted, if necessary, to pH 5 by slowly adding aqueous 1N NaOH. Thesolution is administered using a nebulizer device that provides about 10μg to about 500 μg of the compound of the invention per dose.

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), spray-dried lactose (440 g) andmagnesium stearate (10 g) are thoroughly blended. The resultingcomposition is then loaded into hard gelatin capsules (500 mg ofcomposition per capsule).

Exemplary Suspension for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of compound per 10 mL of suspension:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum ® K(magnesium aluminum silicate) 1.0 g Flavoring 0.035 mL Colorings 0.5 mgDistilled water q.s. to 100 mL

Exemplary Injectable Formulation for Administration by Injection

compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH is then adjusted to pH 4 using 0.5 Naqueous hydrochloric acid or 0.5 N aqueous sodium hydroxide, asnecessary, and then sufficient water for injection is added to provide atotal volume of 20 mL. The mixture is then filtered through a sterilefilter (0.22 micron) to provide a sterile solution suitable foradministration by injection.

Utility

Compounds of the invention possess muscarinic receptor antagonistactivity, and in one embodiment, at nanomolar potencies. In oneembodiment, compounds of the invention are selective for inhibition ofM₃ muscarinic receptor subtype activity over M₂ muscarinic receptorsubtype activity. In another embodiment, compounds of the invention areselective for inhibition of M₃ and M₂ muscarinic receptor subtypeactivity over M₁, M₄, and M₅ muscarinic receptor subtype activity.Additionally, compounds of the invention are expected to possess adesirable duration of action. Accordingly, in another specificembodiment, the invention is directed to compounds having a duration ofaction greater than about 24 hours. Moreover, compounds of the inventionare also expected to possess reduced side effects, such as dry mouth, atefficacious doses when administered by inhalation compared to otherknown muscarinic receptor antagonists administered by inhalation (suchas tiotropium).

One measure of the affinity of a compound for the M₃ receptor subtype isthe inhibition dissociation constant (K_(i)) for binding to thereceptor. Compounds of the invention are expected to have a K_(i) forthe M₃ receptor subtype of less than or equal to 100 nM, as determined,for example, by an in vitro radioligand displacement assay. Compounds ofparticular interest include those having a K_(i) less than or equal to50 nM, and in another embodiment, the compounds have a K_(i) less thanor equal to 10 nM, and in yet another embodiment, the compounds have aK_(i) less than or equal to 1.0 nM. Compounds of even more particularinterest include those having a K_(i) less than or equal to 500 pM, andin another embodiment, the compounds have a K_(i) less than or equal to200 pM. It is noted that in some cases, compounds of the invention maypossess weak muscarinic receptor antagonist activity. In such cases,those of skill in the art will recognize that these compounds still haveutility as research tools.

Also of particular interest are those compounds having an ID₅₀ of lessthan or equal to 100 μg/mL at 24 hours post dosing, more particularlythose compounds having an ID₅₀ of less than or equal to 30 μg/mL at 24hours post dosing.

Exemplary assays to determine properties of compounds of the invention,such as the muscarinic receptor antagonizing activity, are described inthe Examples and include by way of illustration and not limitation,assays that measure hM₁, hM₂, hM₃, hM₄, and hM₅ muscarinic receptorbinding (for example, as described in Assay 1). Useful functional assaysto determine the muscarinic receptor antagonizing activity of compoundsof the invention include by way of illustration and not limitation,assays that measure ligand-mediated changes in intracellular cyclicadenosine monophosphate (cAMP), ligand-mediated changes in activity ofthe enzyme adenylyl cyclase (which synthesizes cAMP), ligand-mediatedchanges in incorporation of guanosine 5′-O-(γ-thio)triphosphate([³⁵S]GTPγS) into isolated membranes via receptor catalyzed exchange of[³⁵S]GTPγS for guanosine diphosphate, ligand-mediated changes in freeintracellular calcium ions (measured, for example, with afluorescence-linked imaging plate reader or FLIPR® from MolecularDevices, Inc.), and the like. Exemplary assays are described in Assay 2.Compounds of this invention are expected to antagonize or decrease theactivation of muscarinic receptors in any of the assays listed above, orassays of a similar nature, and will typically be used in these studiesat a concentration ranging from about 0.1-100 nanomolar. Thus, theaforementioned assays are useful in determining the therapeutic utility,for example, the bronchodilating activity, of compounds of theinvention.

Other properties and utilities of compounds of the invention can bedemonstrated using various in vitro and in vivo assays well-known tothose skilled in the art. For example, the in vivo potency of compoundsof the invention can be measured in an animal model such as theEinthoven model. Briefly, the bronchodilator activity of a compound isevaluated in an anesthetized animal (the Einthoven model), which usesventilation pressure as a surrogate measure of airway resistance. See,for example, Einthoven (1892) Pfugers Arch. 51:367-445; and Mohammed etal. (2000) Pulm Pharmacol Ther. 13(6):287-92, as well as Assay 3 whichdescribes a rat Einthoven model. In one embodiment, a compound of theinvention administered at a dose of 100 μg/ml in the rat Einthoven modelexhibits greater than or equal to 35% inhibition of thebronchoconstrictor response at 24 hours, and in another embodimentexhibits greater than or equal to 70% inhibition at 24 hours. Anotheruseful in vivo assay is the rat antisialagogue assay (for example, asdescribed in Assay 4).

The quaternary compounds of the invention also provide surprisingadvantages over the corresponding non-quaternary compounds, asmanifested, for example, in improved in vivo potency. For example, thefollowing secondary and tertiary compounds:

exhibit a hM₃ K_(i) value of 0.42 and 0.38 nM measured in a bindingassay such as described in Assay 1 (measured at 6 hours), respectively.When evaluated in a rat Einthoven assay such as that described in Assay3 (100 μg dose; measured at 24 hours), the secondary and tertiarycompounds exhibited −6% and −18% inhibition of MCh response relative tocontrol animals, respectively. On the other hand, quaternary compoundsof the invention, such as[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(4-hydroxyphenyl)ethyl]dimethylammonium(Example 3-3):

exhibited a hM₃ K_(i) value of 0.74 nM and 76% inhibition, whenevaluated under the same or similar conditions.

Compounds of the invention are expected to be useful as therapeuticagents for treating medical conditions mediated by muscarinic receptors.Thus it is expected that patients suffering from a disease or disorderthat is treated by blocking the muscarinic receptor can be treated byadministering a therapeutically effective amount of a muscarinicreceptor antagonist of the invention. Such medical conditions include,by way of example, pulmonary disorders or diseases including thoseassociated with reversible airway obstruction, such as chronicobstructive pulmonary disease (e.g., chronic and wheezy bronchitis andemphysema), asthma, pulmonary fibrosis, allergic rhinitis, rhinorrhea,and the like. Other medical conditions that can be treated withmuscarinic receptor antagonists are genitourinary tract disorders, suchas overactive bladder or detrusor hyperactivity and their symptoms;gastrointestinal tract disorders, such as irritable bowel syndrome,diverticular disease, achalasia, gastrointestinal hypermotilitydisorders and diarrhea; cardiac arrhythmias, such as sinus bradycardia;Parkinson's disease; cognitive disorders, such as Alzheimer's disease;dismenorrhea; and the like.

The amount of active agent administered per dose or the total amountadministered per day may be predetermined or it may be determined on anindividual patient basis by taking into consideration numerous factors,including the nature and severity of the patient's condition, thecondition being treated, the age, weight, and general health of thepatient, the tolerance of the patient to the active agent, the route ofadministration, pharmacological considerations such as the activity,efficacy, pharmacokinetics and toxicology profiles of the active agentand any secondary agents being administered, and the like. Treatment ofa patient suffering from a disease or medical condition (such as COPD)can begin with a predetermined dosage or a dosage determined by thetreating physician, and will continue for a period of time necessary toprevent, ameliorate, suppress, or alleviate the symptoms of the diseaseor medical condition. Patients undergoing such treatment will typicallybe monitored on a routine basis to determine the effectiveness oftherapy. For example, in treating COPD, significant improvement inforced expiratory volume (measured in one second) may be used todetermine the effectiveness of treatment. Similar indicators for theother diseases and conditions described herein, are well-known to thoseskilled in the art, and are readily available to the treating physician.Continuous monitoring by the physician will insure that the optimalamount of active agent will be administered at any given time, as wellas facilitating the determination of the duration of treatment. This isof particular value when secondary agents are also being administered,as their selection, dosage, and duration of therapy may also requireadjustment. In this way, the treatment regimen and dosing schedule canbe adjusted over the course of therapy so that the lowest amount ofactive agent that exhibits the desired effectiveness is administeredand, further, that administration is continued only so long as isnecessary to successfully treat the disease or medical condition.

Accordingly, in one embodiment, compounds of the invention are usefulfor treating smooth muscle disorders in mammals, including humans andtheir companion animals (e.g., dogs, cats etc.). Such smooth muscledisorders include, by way of illustration, overactive bladder, chronicobstructive pulmonary disease and irritable bowel syndrome. Typically,suitable doses for treating smooth muscle disorders or other disordersmediated by muscarinic receptors will range from about 0.14 μg/kg/day toabout 7 mg/kg/day of active agent; including from about 0.15 μg/kg/dayto about 5 mg/kg/day. For an average 70 kg human, this would amount toabout 10 μg per day to about 500 mg per day of active agent.

In a specific embodiment, compounds of the invention are useful fortreating pulmonary or respiratory disorders, such as COPD or asthma, inmammals including humans, by administering to a patient atherapeutically effective amount of the compound. Generally, the dosefor treating a pulmonary disorder will range from about 10-1500 μg/day.The term “COPD” is understood by those of ordinary skill in the art toinclude a variety of respiratory conditions, including chronicobstructive bronchitis and emphysema, as exemplified by the teachings ofBarnes (2000) N. Engl. J. Med. 343:269-78, and references cited therein.When used to treat a pulmonary disorder, compounds of the invention areoptionally administered in combination with other therapeutic agentssuch as a β₂-adrenoreceptor agonist; a corticosteroid, a non-steroidalanti-inflammatory agent, or combinations thereof.

When administered by inhalation, compounds of the invention typicallyhave the effect of producing bronchodilation. Accordingly, in another ofits method aspects, the invention is directed to a method of producingbronchodilation in a patient, comprising administering to a patient abronchodilation-producing amount of a compound of the invention.Generally, the therapeutically effective dose for producingbronchodilation will range from about 10-1500 μg/day.

In another embodiment, compounds of the invention are used to treatoveractive bladder. When used to treat overactive bladder, a typicaldose will range from about 1.0-500 mg/day. In yet another embodiment,compounds of the invention are used to treat irritable bowel syndrome.When used to treat irritable bowel syndrome, compounds of the inventionwill typically be administered orally or rectally, and a typical dosewill range from about 1.0-500 mg/day.

Since compounds of this invention possess muscarinic receptor antagonistactivity, such compounds are also useful as research tools forinvestigating or studying biological systems or samples havingmuscarinic receptors. Any suitable biological system or sample havingM₁, M₂, M₃, M₄ and/or M₅ muscarinic receptors may be employed in suchstudies which may be conducted either in vitro or in vivo.Representative biological systems or samples suitable for such studiesinclude, but are not limited to, cells, cellular extracts, plasmamembranes, tissue samples, isolated organs, mammals (such as mice, rats,guinea pigs, rabbits, dogs, pigs, humans, and so forth), and the like,with mammals being of particular interest. In one particular embodimentof the invention a muscarinic receptor in a mammal is antagonized byadministering a muscarinic receptor-antagonizing amount of a compound ofthe invention. Compounds of the invention can also be used as researchtools by conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprising amuscarinic receptor is typically contacted with a muscarinicreceptor-antagonizing amount of a compound of the invention. After thebiological system or sample is exposed to the compound, the effects ofantagonizing the muscarinic receptor are determined using conventionalprocedures and equipment, such as by measuring binding in a radioligandbinding assays or ligand-mediated changes in a functional assay or bydetermining the amount of bronchoprotection provided by the compound ina bronchoprotection assay in a mammal. Exposure encompasses contactingcells or tissue with the compound, administering the compound to amammal, for example by i.p. or i.v. administration, and so forth. Thisdetermining step may comprise measuring a response, i.e., a quantitativeanalysis or may comprise an observation, i.e., a qualitative analysis.Measuring a response involves, for example, determining the effects ofthe compound on the biological system or sample using conventionalprocedures and equipment, such as radioligand binding assays andmeasuring ligand-mediated changes in functional assays. The assayresults can be used to determine the activity level as well as theamount of compound necessary to achieve the desired result, i.e., amuscarinic-antagonizing amount. Typically, the determining step willinvolve determining the muscarinic receptor ligand-mediated effects.

Additionally, compounds of the invention can be used as research toolsfor evaluating other chemical compounds, and thus are also useful inscreening assays to discover, for example, new compounds havingmuscarinic receptor binding activity. In this manner, a compound of theinvention is used as a standard in an assay to allow comparison of theresults obtained with a test compound and with compounds of theinvention to identify those test compounds that have about equal orsuperior binding, if any. For example, muscarinic receptor binding data(as determined, for example, by in vitro radioligand displacementassays) for a test compound or a group of test compounds is compared tothe muscarinic receptor binding data for a compound of the invention toidentify those test compounds that have the desired properties, e.g.,test compounds having binding about equal or superior to a compound ofthe invention, if any. Alternatively, for example, bronchoprotectiveeffects can be determined for test compounds and a compound of theinvention in a bronchoprotection assay in a mammal and this datacompared to identify test compounds providing about equal or superiorbronchoprotective effects. This aspect of the invention includes, asseparate embodiments, both the generation of comparison data (using theappropriate assays) and the analysis of the test data to identify testcompounds of interest. Thus, a test compound can be evaluating in abiological assay, by a method comprising the steps of: (a) conducting abiological assay with a test compound to provide a first assay value;(b) conducting the biological assay with a compound of the invention toprovide a second assay value; wherein step (a) is conducted eitherbefore, after or concurrently with step (b); and (c) comparing the firstassay value from step (a) with the second assay value from step (b).Exemplary biological assays include muscarinic receptor binding assays.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard meaning:

-   -   BSA bovine serum albumin    -   cAMP 3′-5′ cyclic adenosine monophosphate    -   cM₅ cloned chimpanzee M₅ receptor    -   DCM dichloromethane    -   DIPEA N,N-diisopropylethylamine    -   DMA N,N-dimethylacetamide    -   DMF N,N-dimethylformamide    -   dPBS Dulbecco's phosphate buffered saline    -   EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride    -   EDTA ethylenediamine tetraacetic acid    -   EtOAc ethyl acetate    -   HBSS Hank's Buffered Salt Solution    -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   hM₁ cloned human M₁ receptor    -   hM₂ cloned human M₂ receptor    -   hM₃ cloned human M₃ receptor    -   hM₄ cloned human M₄ receptor    -   hM₅ cloned human M₅ receptor    -   HOBt 1-hydroxybenzotriazole hydrate    -   MeOH methanol    -   THF tetrahydrofuran

Any other abbreviations used herein but not defined have their standard,generally accepted meaning. Unless noted otherwise, all materials, suchas reagents, starting materials and solvents, were purchased fromcommercial suppliers (such as Sigma-Aldrich, Fluka Riedel-de Haën, andthe like) and were used without further purification. Reactions were rununder nitrogen atmosphere, unless noted otherwise. Progress of reactionmixtures was monitored by thin layer chromatography (TLC), analyticalhigh performance liquid chromatography (anal. HPLC), and massspectrometry, the details of which are given below and separately inspecific examples of reactions. Reaction mixtures were worked up asdescribed specifically in each reaction; commonly they were purified byextraction and other purification methods such as temperature-, andsolvent-dependent crystallization, and precipitation. In addition,reaction mixtures were routinely purified by preparative HPLC.

Preparation 1 (R)-Cyclopentylhydroxyphenyl acetic Acid

(2R,5R)-2-t-Butyl-5-phenyl-1,3-dioxolan-4-one (1a): (R)-Mandelic acid(20 g, 130 mmol) was dissolved in anhydrous pentane (200 mL, 1.7 mol).Pivaldehyde (13.6 g, 153 mmol) was added followed bytrifluoromethanesulfonic acid (488 μL, 5.4 mmol). The mixture wasallowed to reflux at 36° C. under nitrogen. After 5.5 hours, the mixturewas allowed to cool to room temperature before stirring with 200 mL ofan 8 wt % NaHCO₃ solution for 10 minutes. Excess pentane was removed byrotary evaporation. The solids were collected by filtration and rinsed(100 mL water) while under vacuum filtration. The solids were driedovernight under high vacuum to yield 23.8 grams of intermediate (1a) asa white solid (88% purity).

(2R,5S)-2-t-Butyl-5-(1-hydroxycyclopentyl)-5-phenyl-1,3-dioxolan-4-one(1b): Lithium hexamethyldisilazide (0.8 g, 4.7 mmol; 4.7 mL of 1.0 M inhexanes) was added to anhydrous THF (5.3 mL, 65 mmol) at −78° C.Intermediate (1a) (800 mg, 3.6 mmol) in 5.3 mL anhydrous THF was addedto the solution dropwise over 15 minutes. After 30 minutescyclopentanone (451 μL, 5.1 mmol) was added dropwise over less than 1minute. After 2 hours, 0.8 mL of saturated aqueous Na₂HPO₄ was added,and the mixture stirred at room temperature for 5 minutes. The mixturewas added to 8 mL saturated aqueous ammonium chloride. The aqueous layerwas washed (2×80 mL EtOAc), and the organic layers were combined, driedover Na₂SO₄, filtered, and concentrated. The crude product (780 mg) waspurified by flash chromatography (5-15% EtOAc gradient over 30 minuteswith hexanes) to yield intermediate (1b).

(2R,5S)-2-t-Butyl-5-cyclopent-1-enyl-5-phenyl-1,3-dioxolan-4-one (c):Intermediate (1b) (650 mg, 2.1 mmol) was dissolved in 6.8 mL anhydrousTHF and the solution was cooled to 0° C. Thionyl chloride (436 μL, 6mmol) was added dropwise, followed by the addition of pyridine (777 μL,9.6 mmol). The mixture was stirred at 0° C. for 1 hour. Saturatedaqueous ammonium chloride (14 mL) was added and the mixture was stirredfor 5 minutes while warming to room temperature. The layers wereseparated, and the aqueous layer was washed (2×100 mL EtOAc). Theorganic layers were combined, dried over Na₂SO₄, filtered, andconcentrated to yield intermediate (1c) as a light yellow oil (540 mg),which was used in the next step without further purification.

(S)-Cyclopent-1-enyl-hydroxyphenyl acetic acid (1d): Intermediate (1c)(540 mg, 1.9 mmol) was dissolved in MeOH (927 μL, 22.9 mmol). Water(1.84 mL, 102 mmol) was added, followed by the addition of KOH (1.1 g,18.8 mmol). The mixture was refluxed at 130° C. for 3 hours, followed bydilution to 250 mL with saturated ammonium chloride, then washed (2×100mL hexane). The remaining aqueous emulsion was washed (2×250 mL EtOAc).The EtOAc layers were combined, washed with 50 mL saturated aqueousNaCl, dried over Na₂SO₄, filtered and concentrated to yield intermediate(1d) as a brownish-yellow solid (290 mg).

Intermediate (1d) (280 mg, 1.3 mmol) was dissolved in MeOH (2.50 mL,61.7 mmol) and the reaction flask was flushed with nitrogen before 28 mgof 10% Pd/C was added. The mixture was stirred at room temperature under1 atm hydrogen and the reaction was monitored by HPLC until the startingmaterial was consumed (˜24 hours). The reaction vessel was flushed withnitrogen, then the mixture was filtered through a pad of Celite® andrinsed with MeOH. The filtrate was concentrated under vacuum to obtainthe title compound as a slightly yellow solid (284 mg).

Preparation 2(R)-2-Cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-phenylethanone

To a stirred solution of (R)-cyclopentylhydroxyphenyl acetic acid (5.00g, 22.7 mmol) in DCM (200 mL, 3 mol) was added dimethylpiperidin-4-ylamine (2.91 g, 22.7 mmol). DIPEA (11.9 mL, 68.1 mmol) and HOBt (5.21 g,34 mmol) were added, followed by EDCI (5.22 g, 27.2 mmol). The mixturewas stirred for 12 hours, then washed with water (300 mL), a saturatedaqueous NaCl solution (300 mL), dried over MgSO₄ and then filtered. Thesolvent was removed under reduced pressure. The crude material waspurified via silica gel chromatography (10% MeOH/DCM w/1% NH₃ (aq)) toafford 4.5 g of the title compound.

Example 1[1-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethylphenethylammonium

To a stirred solution of(R)-2-cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-phenylethanone(50 mg, 0.2 mmol) in DMF (1 mL, 10 mmol) was added1-bromo-2-phenylethane (83 mg, 0.5 mmol). The mixture was heated at 80°C. for 24 hours. The solvent was removed under reduced pressure, and thecrude material was purified by preparative HPLC to afford 26.1 mg of thetitle compound as a TFA salt. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O₂, 435.30.found 435.2. ¹H NMR (CD₃OD, 300 MHz) δ (ppm): 1.04 (1H, m), 1.34 (3H,m), 1.56 (6H, m), 1.89 (1H, m), 2.05 (1H, m), 2.55 (1H, m), 2.83 (2H,m), 3.03 (12H, bm), 3.59 (1H, t), 4.66 (1H, m), 7.19 (1H, d), 7.30 (5H,m) 7.37 (2H, t), 7.41 (2H, d).

Preparation 3 3-(2-Bromoethyl)phenol

A solution of 1-(2-bromoethyl)-3-methoxybenzene (1.00 g, 4.7 mmol) inCHCl₃ (50 mL, 0.6 mol) was cooled to at 0° C. 1.0 M of BBr₃ in DCM (27.9mL) was cooled to 0° C. and added to the solution. The solution wasallowed to warm to 30° C. over 30 minutes, then was poured into asolution of ice and 30% NH₄OH, which was stirred at 0° C. for 30minutes. The organic layer was taken and concentrated with no reducedpressure.

Example 2[1-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(3-hydroxyphenyl)ethyl]dimethylammonium

To a stirred solution of(R)-2-cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-phenylethanone(0.8 g, 2.4 mmol) in DMA (10 mL, 0.1 mol) was added3-(2-bromoethyl)phenol (1.5 g, 7.3 mmol). The mixture was heated at 80°C. for 24 hours. The solvent was then removed under reduced pressure.The crude material was purified by preparative HPLC to afford 180 mg ofthe title compound as a TFA salt. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O₃,451.30. found 451.5. ¹H NMR (CD₃OD, 300 MHz) δ (ppm): 1.17 (1H, m), 1.38(3H, m), 1.54 (6H, m), 1.89 (1H, m), 2.02 (1H, m), 2.53 (1H, m), 2.79(2H, m), 2.95 (12H, bm), 3.56 (1H, t), 4.66 (1H, m), 6.70 (3H, m), 7.14(1H, t) 7.16 (1H, d) 7.29 (2H, t), 7.42 (2H, d).

Example 3

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds3-1 to 3-24, having the following formula, were also prepared:

Ex. R^(Q) Name 3-1 3-fluoro[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(3-fluorophenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₈FN₂O₂, 453.29; found 453.4. 3-2 4-fluoro[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(4-fluorophenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₈FN₂O₂, 453.29; found 453.4. 3-3 4-hydroxy[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(4-hydroxyphenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₉N₂O₃, 451.30; found 451.4. 3-4 absent[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethylphenethylammonium. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O₂, 435.30;found 435.4. 3-5 2-fluoro[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(2-fluorophenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₈FN₂O₂, 453.29; found 453.4. 3-6 2,4-dichloro[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(2,4-dichlorophenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₇Cl₂N₂O₂, 503.22; found 503.2. 3-7 2-chloro[2-(2-chlorophenyl)ethyl]-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₈ClN₂O₂, 469.26; found 469.2. 3-8 2-hydroxy-ethyl[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]-{2-[4-(2-hydroxyethyl)phenyl]ethyl}dimethyl-ammonium. MS m/z: [M⁺] calcdfor C₃₀H₄₃N₂O₃, 479.33; found 479.4. 3-9 2-chloro-4-fluoro[2-(2-chloro-4-fluorophenyl)ethyl][1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl) piperidin-4-yl]dimethyl-ammonium. MS m/z: [M⁺] calcd forC₂₈H₃₇ClFN₂O₂, 487.25; found 487.2 3-10 4-methyl[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethyl-(2-p-tolylethyl)ammonium. MS m/z: [M⁺] calcd for C₂₉H₄₁N₂O₂,449.32; found 449.4. 3-11 4-bromo[2-(4-bromophenyl)ethyl][1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₈BrN₂O₂, 513.21; found 513.2. 3-12 4-cyano[2-(4-cyanophenyl)ethyl][1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₉H₃₈N₃O₂, 460.30; found 460.4. 3-13 4-hydroxy-3-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2- methoxy(4-hydroxy-3-methoxyphenyl)ethyl]dimethyl-ammonium. MS m/z: [M⁺] calcdfor C₂₉H₄₁N₂O₃, 481.31; found 481.4. 3-14 3-methoxy[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(3-methoxyphenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₉H₄₁N₂O₃, 465.31; found 465.4. 3-15 2-bromo[2-(2-bromophenyl)ethyl][1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₈BrN₂O₂, 513.21; found 513.2. 3-16 4-methoxy[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(4-methoxyphenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₉H₄₁N₂O₃, 465.31; found 465.4. 3-17 absent[(R)-1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)pyrrolidin-3-yl]dimethylphenethylammonium. MS m/z: [M⁺] calcd for C₂₇H₃₇N₂O₂, 421.29;found 421.6. 3-18 4-hydroxy[1-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]-[2-(4-hydroxyphenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₉N₂O₃, 451.30; found 451.2. 3-19 4-hydroxy[1-((S)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(4-hydroxyphenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₉N₂O₃, 451.30; found 451.2. 3-20 2-fluoro-4-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2- methoxy(2-fluoro-4-methoxyphenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₉H₄₀FN₂O₃, 483.30; found 483.2. 3-21 2-fluoro-4-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2- hydroxy(2-fluoro-4-hydroxyphenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₈FN₂O₃, 469.29; found 469.2. 3-22 3-hydroxy[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(3-hydroxyphenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₉N₂O₃, 451.30; found 451.2. 3-23 3-fluoro[1-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(3-fluorophenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₈FN₂O₂, 453.29; found 453.2. 3-24 2-hydroxy[1-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(2-hydroxyphenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₉N₂O₃, 451.30; found 451.2.

Example 4

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds4-1 to 4-8, having the following formula, were also prepared:

Ex. Q Name 4-1 1H-indo1-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin- 3-yl4-yl][2-(1H-indol-3-yl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₃₀H₄₀N₃O₂, 474.31; found 474.4. 4-2 thiophen-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin- 2-yl4-yl]dimethyl(2-thiophen-2-ylethyl)ammonium. MS m/z: [M⁺] calcd forC₂₆H₃₇N₂O₂S, 441.26; found 441.2. 4-3 benzo[1,3]di-(2-benzo[1,3]dioxol-5-ylethyl)[1-(2-cyclopentyl-2- oxol-5-ylhydroxy-2-phenylacetyl)piperidin-4-yl]dimethyl- ammonium. MS m/z: [M⁺]calcd for C₂₉H₃₉N₂O₄, 479.29; found 479.4. 4-4 thiophen-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin- 3-yl4-yl]dimethyl(2-thiophen-3-ylethyl)ammonium. MS m/z: [M⁺] calcd forC₂₆H₃₇N₂O₂S, 441.26; found 441.2. 4-5 pyrrol-1-yl[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethyl(2-pyrrol-1-ylethyl)ammonium. MS m/z: [M⁺] calcd forC₂₆H₃₈N₃O₂, 424.30; found 424.4. 4-6 1H-tetrazol-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin- 5-yl4-yl]dimethyl-[2-(1H-tetrazol-5-yl)ethyl]ammonium. MS m/z: [M⁺] calcdfor C₂₃H₃₅N₆O₂, 427.28; found 427.2. 4-7 thiophen-[1-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piper- 3-ylidin-4-yl]dimethyl-(2-thiophen-3-ylethyl)ammonium. MS m/z: [M⁺] calcdfor C₂₆H₃₇N₂O₂S, 441.26; found 441.2. 4-8 thiophen-[1-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piper- 2-ylidin-4-yl]dimethyl(2-thiophen-2-ylethyl)ammonium. MS m/z: [M⁺] calcd forC₂₆H₃₇N₂O₂S, 441.26; found 441.2.

Example 5

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds5-1 to 5-3, having the following formula, were also prepared:

Ex. R Name 5-1 phenyl(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]-(3,3-diphenylpropyl)dimethylammonium. MS m/z: [M⁺] calcd for C₃₅H₄₅N₂O₂,525.35; found 525.4. 5-2 —OH[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]-((R)-2-hydroxy-2-phenylethyl)dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₉N₂O₃, 451.30; found 451.2. 5-3 —OH[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]-((S)-2-hydroxy-2-phenylethyl)dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₉N₂O₃, 451.30; found 451.2.

Example 6

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds6-1 to 6-14, having the following formula, were also prepared:

Ex. R^(Q) Name 6-1 2-methoxy-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl](2- 4-methoxy-methoxy-4-methoxycarbonylbenzyl)dimethylammonium. MS m/z: carbonyl [M⁺]calcd for C₃₀H₄₁N₂O₅, 509.30; found 509.4. 6-2 4-trifluoro-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethyl-methoxy (4-trifluoromethoxybenzyl)ammonium. MS m/z: [M⁺] calcd forC₂₈H₃₆F₃N₂O₃, 505.27; found 505.4. 6-3 4-chloro(4-chlorobenzyl)-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piper-idin-4-yl]dimethylammonium. MS m/z: [M⁺] calcd for C₂₇H₃₆ClN₂O₂, 455.25;found 455.2. 6-4 3-fluoro[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl](3-fluoro-benzyl)dimethylammonium. MS m/z: [M⁺] calcd for C₂₇H₃₆FN₂O₂, 439.28;found 439.4. 6-5 3-nitro[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimeth-yl(3-nitrobenzyl)ammonium. MS m/z: [M⁺] calcd for C₂₇H₃₆N₃O₄, 466.27;found 466.4. 6-6 4-t-butyl(4-t-butylbenzyl)[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]-dimethylammonium. MS m/z: [M⁺] calcd for C₃₁H₄₅N₂O₂, 477.35; found477.4. 6-7 4-trifluoro-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethyl(4-methyl trifluoromethylbenzyl)ammonium. MS m/z: [M⁺] calcd forC₂₈H₃₆F₃N₂O₂, 489.27; found 489.4. 6-8 4-bromo(4-bromobenzyl)[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethylammonium. MS m/z: [M⁺] calcd for C₂₇H₃₆BrN₂O₂, 499.20;found 499.2. 6-9 4-cyano(4-cyanobenzyl)-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethylammonium. MS m/z: [M⁺] calcd for C₂₈H₃₆N₃O₂, 446.28; found446.4. 6-10 4-fluoro[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]-(4-fluoro-benzyl)dimethylammonium. MS m/z: [M+] calcd for C₂₇H₃₆FN₂O₂, 439.28;found 439.4. 6-11 4-methyl[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethyl(4-methylbenzyl)ammonium. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O₂, 435.30; found435.4. 6-12 3-methyl[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethyl(3-methylbenzyl)ammonium. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O₂, 435.30; found435.4. 6-13 4-nitro[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethyl(4-nitrobenzyl)ammonium. MS m/z: [M⁺] calcd for C₂₇H₃₆N₃O₄, 466.27; found466.4. 6-14 4-carboxy-(4-carboxymethylbenzyl)[1-(2-cyclopentyl-2-hydroxy-2-phenylacet- methylyl)piperidin-4-yl]dimethylammonium. MS m/z: [M⁺] calcd for C₂₉H₃₉N₂O₄,479.29; found 479.4.

Example 7

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds7-1 to 7-11, having the following formula, were also prepared:

Ex. R^(Q) Name 7-1 absent[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl]piper-idin-4-yl]dimethyl(2-oxo-2-phenylethyl)ammonium. MS m/z: [M⁺] calcd forC₂₈H₃₇N₂O₃, 449.28; found 449.3. 7-2 4-bromo[2-(4-bromophenyl)-2-oxoethyl]-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimeth- ylammonium. MS m/z: [M⁺]calcd for C₂₈H₃₆BrN₂O₃, 528.19; found 529.2. 7-3 4-methoxy[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piper-idin-4-yl]-[2-(4-methoxyphenyl)-2-oxoethyl]dimeth- ylammonium. MS m/z:[M⁺] calcd for C₂₉H₃₉N₂O₄, 479.29; found 479.2. 7-4 3-cyano[2-(3-cyanophenyl)-2-oxoethyl][1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimeth- ylammonium. MS m/z: [M⁺]calcd for C₂₉H₃₆N₃O₃, 474.28; found 474.2. 7-5 4-cyano[2-(4-cyanophenyl)-2-oxoethyl][1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimeth- ylammonium. MS m/z: [M⁺]calcd for C₂₉H₃₆N₃O₃, 474.28; found 474.2. 7-6 4-chloro[2-(4-chlorophenyl)-2-oxoethyl][1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimeth- ylammonium. MS m/z: [M⁺]calcd for C₂₈H₃₆ClN₂O₃, 483.24; found 483.2. 7-7 3-fluoro[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piper-idin-4-yl][2-(3-fluorophenyl)-2-oxoethyl]dimeth- ylammonium. MS m/z:[M⁺] calcd for C₂₈H₃₆FN₂O₃, 467.27; found 467.2. 7-8 2-methoxy[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piper-idin-4-yl][2-(2-methoxyphenyl)-2-oxoethyl]dimeth- ylammonium. MS m/z:[M⁺] calcd for C₂₉H₃₉N₂O₄, 479.29; found 479.2. 7-9 4-difluoro-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piper- methoxyidin-4-yl][2-(4-difluoromethoxyphenyl)-2-oxoeth- yl]dimethyl-ammonium.MS m/z: [M⁺] calcd for C₂₉H₃₇F₂N₂O₄, 515.27; found 515.2. 7-10 3-methoxy[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piper-idin-4-yl][2-(3-methoxyphenyl)-2-oxoethyl]dimeth- ylammonium.MS m/z:[M⁺] calcd for C₂₉H₃₉N₂O₄, 479.29; found 479.2. 7-11 2-chloro[2-(2-chlorophenyl)-2-oxoethyl][1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimeth- ylammonium. MS m/z: [M⁺]calcd for C₂₈H₃₆ClN₂O₃, 483.24; found 483.2.

Example 8

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds8-1 to 8-4, having the following formula, were also prepared:

Ex. a R^(Q) Name 8-1 2 ab-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4- sentyl]dimethyl-(2-phenoxyethyl)ammonium. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O₃,451.30; found 451.4. 8-2 4 ab-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4- sentyl]dimethyl-(4-phenoxybutyl)ammonium. MS m/z: [M⁺] calcd for C₃₀H₄₃N₂O₃,479.33; found 479.4. 8-3 3 ab-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4- sentyl]dimethyl-(3-phenoxypropyl)ammonium. MS m/z: [M⁺] calcd forC₂₉H₄₁N₂O₃, 465.31; found 465.4. 8-4 2 4-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4- fluoroyl][2-(4-fluorophenoxy)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₈FN₂O₃, 469.29; found 469.4.

Example 9

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds9-1 to 9-10, having the following formula, were also prepared:

Ex. a b R^(Q) Name 9-1 1 1 absentbenzyl-[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)-piperidin-4-ylmethyl]dimethylammonium. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O₂, 435.30;found 435.2. 9-2 1 2 absent[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-ylmeth-yl]dimethylphenethylammonium. MS m/z: [M⁺] calcd for C₂₉H₄₁N₂O₂, 449.32;found 449.2. 9-3 1 3 absent[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-ylmeth-yl]dimethyl(3-phenylpropyl)ammonium. MS m/z: [M⁺] calcd for C₃₀H₄₃N₂O₂,463.33; found 463.2. 9-4 1 1 3-methyl[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-ylmeth-yl]dimethyl(3-methylbenzyl)ammonium. MS m/z: [M⁺] calcd for C₂₉H₄₁N₂O₂,449.32; found 449.2. 9-5 1 1 2-hydroxy[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-ylmeth-yl](2-hydroxybenzyl)dimethylammonium. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O₃,451.30; found 451.2. 9-6 1 1 3-hydroxy[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-ylmeth-yl](3-hydroxybenzyl)dimethylammonium. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O₃,451.30; found 451.0. 9-7 1 1 4-hydroxy[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-ylmeth-yl]-4-hydroxybenzyl)dimethylammonium. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O₃,451.30; found 451.0. 9-8 0 3 absent[(R)-1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)-pyrrolidin-3-yl]dimethyl(3-phenylpropyl)ammonium. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O₂,435.30; found 435.2. 9-9 0 2 4-carboxy[2-(4-carboxyphenyl)ethyl][1-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethyl-ammonium. MS m/z: [M⁺] calcd forC₂₉H₃₉N₂O₄, 479.29. 9-10 0 2 4-methoxy-[1-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]- carbonyl[2-(4-methoxycarbonylphenyl)ethyl]dimethyl-ammonium. MS m/z: [M⁺] calcdfor C₃₀H₄₁N₂O₄, 493.31.

Example 10

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds10-1 to 10-6, having the following formula, were also prepared:

Ex. R⁶ Name 10-1 —CH₂CH₃[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]ethyl-[2-(4-hydroxyphenyl]ethyl]methylammonium. MS m/z: [M⁺] calcdfor C₂₉H₄₁N₂O₃, 465.31; found 465.2. 10-2 —(CH₂)₂CH₃[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(4-hydroxyphenyl)ethyl]methylpropylammonium. MS m/z: [M⁺] calcdfor C₃₀H₄₃N₂O₃, 479.33; found 479.4. 10-3 —CH₂-cyclopropyl[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]cyclopropylmethyl[2-(4-hydroxyphenyl)ethyl]methyl- ammonium. MS m/z:[M⁺] calcd for C₃₁H₄₃N₂O₃, 491.33; found 491.4. 10-4 —(CH₂)₂OH[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl](2-hydroxyethyl)[2-(4-hydroxyphenyl)ethyl]methyl-ammonium. MS m/z: [M⁺]calcd for C₂₉H₄₁N₂O₄, 481.31; found 481.4. 10-5 —CH₂—C(O)OCH₃[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(4-hydroxyphenyl)ethyl]methoxycarbonylmethylmethyl- ammonium. MS m/z:[M⁺] calcd for C₃₀H₄₁N₂O₅, 509.30; found 509.2. 10-6 —CH₂—C(O)NH₂carbamoylmethyl[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(4-hydroxyphenyl)ethyl] methylammonium.MS m/z: [M⁺] calcd for C₂₉H₄₀N₃O₄, 494.30; found 494.2.

Example 11

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds11-1 to 11-3, having the following formula, were also prepared:

Ex. b Name 11-1 2[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)azetidin-3-yl]dimethylphenethylammonium. MS m/z: [M⁺] calcd for C₂₆H₃₅N₂O₂, 407.27;found 407.2. 11-2 3[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)azetidin-3-yl]dimethyl(3-phenylpropyl)ammonium. MS m/z: [M⁺] calcd for C₂₇H₃₇N₂O₂,421.29; found 421.2. 11-3 4[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)azetidin-3-yl]dimethyl(4-phenylbutyl)ammonium. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O₂,435.30; found 435.4.

Example 12

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, thefollowing compound was also prepared:

[1-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl]dimethyl(2-pyrazol-1-ylethyl)ammonium.MS m/z: [M⁺] calcd for C₂₅H₃₇N₄O₂, 425.29. found 425.6.

Example 13[1-((R)-2-Cyclopentyl-2-hydroxy-2-phenylacetyl)piperidin-4-yl][2-(2-fluorophenyl)ethyl]dimethylammonium(13-1) and[1-(2-Cyclopentylidene-2-phenylacetyl)piperidin-4-yl]-[2-(2-fluorophenyl)ethyl]dimethylammonium(13-2)

To a stirred solution of(R)-2-cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-phenylethanone(50 mg, 0.2 mmol) in DMF (1 mL, 10 mmol), was added1-(2-bromoethyl)-2-fluorobenzene (91 mg, 0.5 mmol). The mixture washeated at 80° C. for 24 hours. The solvent was removed under reducedpressure, and the crude material was purified by preparative HPLC toafford 12.2 mg of compound (13-1) as a TFA salt. Additionally theeliminated compound was obtained during the preparative HPLC to afford18.6 mg of compound (13-2).

(13-1) MS m/z: [M⁺] calcd for C₂₈H₃₈FN₂O₂, 453.29. found 453.2.

(13-2) MS m/z: [M⁺] calcd for C₂₈H₃₆FN₂O, 435.28. found 435.4.

Preparation 42-Cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-thiophen-2-ylethanone

1-(4-Dimethylaminopiperidin-1-yl)-2-thiophen-2-yl-ethane-1,2-dione (4a):2-Thiopheneglyoxylic acid (300 mg, 1.9 mmol) was dissolved in 10 mL ofmethylene chloride. A 2.0 M solution of oxalyl chloride in methylenechloride (1.1 mL, 2.1 mmol) was then added, followed by 10 μL of DMF.The resulting solution was stirred at room temperature for 1 hour, thena solution of dimethylpiperidin-4-ylamine (271 mg, 2.1 mmol) and DIPEA(1.0 mL, 5.8 mmol) in 2 mL of methylene chloride was added.4-Dimethylaminopyridine (12 mg, 96 μmol) was then added and theresulting mixture was stirred at room temperature for 2 hours. Themixture was then extracted with a saturated sodium bicarbonate solution,and the organic layer was dried over Na₂SO₄ and concentrated to yieldthe crude intermediate (4a) (498 mg), which was used in the next stepwithout further purification.

Intermediate (4a) (498 mg, 1.87 mmol) was dissolved in 10 mL of THF, andthe solution was cooled to 0° C. A 2.0 M solution of cyclopentylmagnesium bromide in ether (1.12 mL, 2.24 mmol) was added slowly and themixture was stirred at 0° C. for 4 hours. The mixture was then quenchedby the addition saturated ammonium chloride solution. The resultingmixture was extracted with EtOAc, then the organic layer was dried overNa₂SO₄ and concentrated to yield the crude title compound (406 mg),which was used in the next step without further purification.

Example 14[1-(2-Cyclopentyl-2-hydroxy-2-thiophen-2-yl-acetyl)piperidin-4-yl]-[2-(4-hydroxyphenyl)ethyl]dimethylammonium

(2-Cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-thiophen-2-ylethanone(75 mg, 220 μmol) was dissolved in 1 mL of DMF. DIPEA (104 μL, 594 μmol)and 4-(2-bromoethyl)phenol (89.6 mg, 446 μmol) were then added and themixture was stirred at 60° C. for 8 hours. The mixture was thenconcentrated, after which the residue was dissolved in a 1:1 mixture ofwater and acetonitrile and purified by liquid chromatography. 1.6 mg ofthe trifluoroacetate salt of the title compound was isolated as a whitepowder. MS m/z: [M⁺] calcd for C₂₆H₃₇N₂O₃S; 457.25 found 457.2.

Example 15

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds15-1 to 15-3, having the following formula, were also prepared:

Ex. R^(Q) Name 15-1 absent[1-(2-cyclopentyl-2-hydroxy-2-thiophen-2-ylacetyl)piperidin-4-yl]dimethylphenethylammonium. MS m/z: [M⁺] calcd for C₂₆H₃₇N₂O₂S,441.26; found 441.5. 15-2 3-[1-(2-cyclopentyl-2-hydroxy-2-thiophen-2-ylacetyl)piperidin- fluoro4-yl]-[2-(3-fluorophenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₆H₃₆FN₂O₂S, 459.25; found 459.2. 15-3 4-[1-(2-cyclopentyl-2-hydroxy-2-thiophen-2-ylacetyl)piperidin- fluoro4-yl]-[2-(4-fluorophenyl)ethyl]dimethylammonium. MS m/z: [M⁺] calcd forC₂₆H₃₆FN₂O₂S, 459.25; found 459.2.

Preparation 51-(4-Dimethylamino-piperidin-1-yl)-2-hydroxy-4-methyl-2-phenylpentan-1-one

1-(4-Dimethylamino-piperidin-1-yl)-2-phenylethane-1,2-dione (5a):Benzoylformic acid (1.0 g, 6.7 mmol), dimethylpiperidin-4-ylamine (854mg, 6.7 mmol), DIPEA (3.5 mL, 20.0 mmol), and 1-hydroxybenzotriazole(1.4 g, 10 mmol) were dissolved in 20 mL of methylene chloride, thenN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.53 g,7.99 mmol) was added and the mixture was stirred at room temperature for16 hours. The mixture was extracted with a 1.0 N NaOH solution, then theorganic layer was dried over Na₂SO₄ and concentrated. The crude productwas purified by column chromatography (10-30% MeOH in methylene chloridegradient) to give intermediate (5a) (501 mg).

Intermediate (5a) (75.0 mg, 288 μmol) was dissolved in 2 mL of THF, andthe solution was cooled to 0° C. A 2.0 M solution of isobutyl magnesiumbromide in ether (158 μL, 317 μmol) was added slowly and the mixture wasstirred at 0° C. for 4 hours. The mixture was then quenched by theaddition of a saturated sodium bicarbonate solution. The resultingmixture was extracted with EtOAc, then the organic layer was dried overNa₂SO₄ and concentrated to give the crude title compound, which was usedin the next step without further purification.

Example 16[1-(2-Hydroxy-4-methyl-2-phenylpentanoyl)piperidin-4-yl]dimethylphenethylammonium

1-(4-dimethylamino-piperidin-1-yl)-2-hydroxy-4-methyl-2-phenylpentan-1-onewas dissolved in 1 mL of DMF and 1-bromo-2-phenylethane (43.3 μL, 317μmol) was added. The mixture was then microwaved (140° C., 300 watts, 7minutes), after which the mixture was concentrated. The residue was thendissolved in a 1:1 mixture of water and acetonitrile and purified byliquid chromatography. 4.7 mg of the trifluoroacetate salt of the titlecompound was isolated as a white powder. MS m/z: [M⁺] calcd forC₂₇H₃₉N₂O₂, 423.30. found 423.2.

Example 17

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds17-1 and 17-2, having the following formula, were also prepared:

Ex. R¹ R³ Name 17-1 cyclopropyl —OH[1-(2-cyclopropyl-2-hydroxy-2-phenylacetyl)-piperidin-4-yl]dimethylphenethylammonium. MS m/z: [M⁺] calcd forC₂₆H₃₅N₂O₂, 407.27; found 407.2. 17-2 —CH₂—CH═CH₂ —OH[1-(2-hydroxy-2-phenyl-pent-4-enoyl)piperidin-4-yl]dimethylphenethylammonium. MS m/z: [M⁺] calcd for C₂₆H₃₅N₂O₂, 407.27;found 407.2. 17-3 cyclopropyl —CH₂—OH[1-(2-cyclopentyl-3-hydroxy-2-phenylpropionyl)-piperidin-4-yl]dimethylphenethylammonium. MS m/z: [M⁺] calcd forC₂₉H₄₁N₂O₂, 450.32; found 449.2. 17-4 —(CH₂)₂—SCH₃ Hdimethyl-[1-(4-methylsulfanyl-2-phenylbutyryl)-piperidin-4-yl]phenethylammonium. MS m/z: [M⁺] calcd for C₂₆H₃₇N₂OS,426.26; found 426.5. 17-5 —(CH₂)₂—SCH₃ —CH₂—OH[1-(2-hydroxymethyl-4-methylsulfanyl-2-phenylbutyryl)piperidin-4-yl]dimethylphenethyl- ammonium. MS m/z: [M⁺]calcd for C₂₇H₃₉N₂O₂S, 456.27; found 456.2. 17-6 —C≡CH —OH[1-(2-hydroxy-2-phenylbut-3-ynoyl)piperidin-4-yl]dimethylphenethylammonium. MS m/z: [M⁺] calcd for C₂₅H₃₁N₂O₂, 392.24;found 391.5.

Preparation 61-(4-Dimethylamino-piperidin-1-yl)-2-hydroxy-2,2-di-thiophen-2-ylethanone

To a stirred solution of 2-thiopheneglyoxylic acid (1.0 g, 6.4 mmol) inDCM (100 mL, 2 mol), was added oxalyl chloride (596 μL, 7 mmol) and acatalytic amount of DMF. The mixture was stirred for 2 hours and thenwas cooled at 0° C. in an ice bath. Dimethylpiperidin-4-ylamine (821 mg,6.4 mmol), DIPEA (1.7 mL, 9.6 mmol) and DMAP (20 mg, 0.1 mmol) wereadded, and the mixture was stirred for 2 hours. The solvent was removedunder reduced pressure and the crude mixture was then dissolved in DCM(60 mL), washed with a saturated bicarbonate solution (50 mL), and driedover MgSO₄. The solvent was removed under reduced pressure and the crudematerial was then dissolved in THF (50 mL, 0.6 mol) and cooled to 0° C.in an ice bath. To the stirred solution was added 1.0 M thiophen-2-ylmagnesium bromide in THF (7.7 mL, 7.7 mmol) was added. The mixture wasstirred for 30 minutes, then quenched with a saturated bicarbonatesolution (50 mL) and DCM (50 mL). The organic was taken and dried overMgSO₄, and the solvent was removed under reduced pressure, to afford thetitle compound (1.5 g).

MS m/z: [M⁺] calcd for C₁₇H₂₂N₂O₂S₂, 350.50. found 351.4.

Example 18[2-(2-Fluorophenyl)ethyl]-[1-(2-hydroxy-2,2-di-thiophen-2-ylacetyl)piperidin-4-yl]dimethylammonium

The title compound was prepared using the procedure described in Example13, and replacing(R)-2-cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-phenylethanonewith1-(4-dimethylamino-piperidin-1-yl)-2-hydroxy-2,2-di-thiophen-2-ylethanone.MS m/z: [M⁺] calcd for C₂₅H₃₀FN₂O₂S₂, 473.17. found 473.2.

Example 19

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds19-1 to 19-4, having the following formula, were also prepared:

Ex. Q Name 19-1

[1-(2-hydroxy-2,2-dithiophen-2-yl-acetyl)piperidin-4-yl]dimethylphenethylammonium. MS m/z: [M⁺] calcd for C₂₅H₃₁N₂O₂S₂, 455.18;found 455.2. 19-2

(2-benzo[1,3]dioxol-5-yl-ethyl)-[1-(2-hydroxy-2,2-dithiophen-2-ylacetyl)piperidin-4-yl]dimethyl-ammonium. MS m/z: [M⁺]calcd for C₂₆H₃₁N₂O₄S₂, 499.17; found 499.2. 19-3

[1-(2-hydroxy-2,2-dithiophen-2-yl-acetyl)piperidin-4-yl]dimethyl(2-m-tolylethyl)ammonium. MS m/z: [M⁺] calcd for C₂₆H₃₃N₂O₂S₂,469.20; found 469.2. 19-4

[1-(2-hydroxy-2,2-dithiophen-2-yl-acetyl)piperidin-4-yl]dimethyl(2-p-tolylethyl)ammonium. MS m/z: [M⁺] calcd for C₂₆H₃₃N₂O₂S₂,469.20; found 469.2.

Example 20[1-(2-Cyclopentyl-2-phenylacetyl)piperidin-4-yl]dimethylphenethylammonium

The title compound was synthesized using the procedure described inExample 1 and using2-cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-phenylethanone insteadof(R)-2-cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-phenylethanone.2-cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-phenylethanone wasprepared as described in Preparation 2, using α-cyclopentylphenyl aceticacid instead of (R)-cyclopentylhydroxyphenyl acetic acid as the startingmaterial. MS m/z: [M⁺] calcd for C₂₈H₃₉N₂O, 419.31. found 419.2.

Preparation 72-Cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-p-tolylethanone

The title compound was synthesized using the procedure described inPreparation 6, and replacing 2-thiopheneglyoxylic acid with(4-methylphenyl)(oxo)acetic acid, and replacing 1.0 M thiophen-2-ylmagnesium bromide in THF with 2.0 M cyclopentyl magnesium bromide inether.

Example 21[1-(2-Cyclopentyl-2-hydroxy-2-p-tolylacetyl)piperidin-4-yl]dimethylphenethylammonium

The title compound was synthesized using the procedure described inExample 13, and replacing(R)-2-cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-phenylethanonewith2-cyclopentyl-1-(4-dimethylaminopiperidin-1-yl)-2-hydroxy-2-p-tolylethanone.MS m/z: [M⁺] calcd for C₂₉H₄₁N₂O₂, 449.32. found 449.2

Example 22

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds22-1 to 22-5, having the following formula, were also prepared:

Ex. R² Name 22-1

{1-[2-cyclopentyl-2-hydroxy-2-(3-hydroxyphenyl)-acetyl]piperidin-4-yl}dimethylphenethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₉N₂O₃, 451.30; found 451.2. 22-2

{1-[2-cyclopentyl-2-(3-fluoro-4-methoxyphenyl)-2-hydroxy-acetyl]-piperidin-4-yl}dimethylphenethyl-ammonium. MS m/z: [M⁺] calcdfor C₂₉H₄₀FN₂O₃, 483.30; found 483.2. 22-3

{1-[2-cyclopentyl-2-(4-fluorophenyl)-2-hydroxy-acetyl]piperidin-4-yl}dimethyl-phenethylammonium. MS m/z: [M⁺] calcd forC₂₈H₃₈FN₂O₂, 453.29; found 453.2. 22-4

[1-(2-cyclopentyl-2-hydroxy-2-m-tolylacetyl)-piperidin-4-yl]dimethylphenethylammonium. MS m/z: [M⁺] calcd for C₂₉H₄₁N₂O₂, 449.32;found 449.2. 22-5

[1-(2-cyclopentyl-2-hydroxy-2-o-tolylacetyl)-piperidin-4-yl]dimethylphenethylammonium. MS m/z: [M⁺] calcd for C₂₉H₄₁N₂O₂, 449.32;found 449.2.

Preparation 8 (4-Dimethylaminopiperidin-1-yl)(9H-xanthen-9-yl)methanone

To a stirred solution of 9H-xanthene-9-carboxylic acid (4.4 g, 19.4mmol) in DCM (200 mL, 3 mol) was added dimethylpiperidin-4-yl-amine (2.5g, 19.4 mmol). DIPEA (6.8 mL, 38.9 mmol) and HOBt (4.6 g, 34 mmol) wereadded to the mixture, followed by EDCI (4.5 g, 23.3 mmol). The mixturewas stirred for 12 hours, then washed with water (300 mL), NaCl (sat.)(300 mL), dried over MgSO₄ and then filtered. The solvent was removedunder reduced pressure, and the crude material was purified via silicagel chromatography (10% MeOH/DCM w/1% NH₃ (aq)) to yield the titlecompound (5.9 g).

Example 23[2-(2-Fluorophenyl)ethyl]dimethyl-[1-(9H-xanthene-9-carbonyl)piperidin-4-yl]ammonium

To a stirred solution of(4-dimethylaminopiperidin-1-yl)(9H-xanthen-9-yl)methanone (50 mg; 0.2mmol) in DMF (1 mL, 10 mmol) was added 1-(2-bromoethyl)-2-fluorobenzene(30 mg, 0.2 mmol). The mixture was heated at 80° C. for 24 hours. Thesolvent was removed under reduced pressure, and the crude material waspurified by preparative HPLC to yield the title compound as a TFA salt(36.7 mg). MS m/z: [M⁺] calcd for C₂₉H₃₂FN₂O₂, 460.24. found 459.2.

Example 24

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds24-1 to 24-4, having the following formula, were also prepared:

Ex. b Z Q Name 24-1 2 bond

[2-(4-hydroxyphenyl)ethyl]dimethyl-[1-(9H-xanthene-9-carbonyl)piperidin-4- yl]ammonium. MS m/z: [M+] calcd forC₂₉H₃₃N₂O₃, 458.25; found 457.2. 24-2 2 bond

dimethyl-(2-thiophen-3-ylethyl)-[1-(9H- xanthene-9-carbonyl)piperidin-4-yl]ammonium. MS m/z: [M⁺] calcd for C₂₇H₃₁N₂O₂S, 448.21; found 447.2.24-3 1 —C(O)—

dimethyl-(2-oxo-2-phenylethyl)-[1-(9H- xanthene-9-carbonyl)piperidin-4-yl]ammonium. MS m/z: [M⁺] calcd for C₂₉H₃₁N₂O₃, 456.23; found 455.2.24-4 2 bond

dimethylphenethyl-[1-(9H-xanthene-9- carbonyl)piperidin-4-yl]ammonium.MS m/z: [M⁺] calcd for C₂₉H₃₃N₂O₂, 442.25; found 441.2.

Example 25

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds25-1 to 25-3, having the following formula, were also prepared:

Ex. b Name 24-1 2 dimethylphenethyl-[1-(9H-xanthene-9-carbonyl)azetidin-3-yl]ammonium. MS m/z: [M⁺] calcd for C₂₇H₂₉N₂O₂,414.22; found 413.2. 24-2 3 dimethyl-(3-phenylpropyl)-[1-(9H-xanthene-9-carbonyl)azetidin-3-yl]ammonium. MS m/z: [M⁺] calcd for C₂₈H₃₁N₂O₂,428.24; found 427.2. 24-3 4 dimethyl-(4-phenylbutyl)-[1-(9H-xanthene-9-carbonyl)azetidin-3-yl]ammonium. MS m/z: [M⁺] calcd for C₂₉H₃₃N₂O₂,442.25; found 441.2.

Assay 1 Radioligand Binding Assay Membrane Preparation from CellsExpressing hM₁, hM₂, hM₃ and hM₄ Muscarinic Receptor Subtypes

Chinese hamster ovary (CHO) cell lines stably expressing cloned humanhM₁, hM₂, hM₃ and hM₄ muscarinic receptor subtypes, respectively, weregrown to near confluency in medium consisting of HAM's F-12 supplementedwith 10% fetal bovine serum and 250 g/mL Geneticin. The cells were grownin a 5% CO₂, 37° C. incubator and lifted with 2 mM EDTA in dPBS. Cellswere collected by 5 minute centrifugation at 650×g, and cell pelletswere either stored frozen at −80° C. or membranes were preparedimmediately. For membrane preparation, cell pellets were resuspended inlysis buffer and homogenized with a Polytron PT-2100 tissue disrupter(Kinematica AG; 20 seconds×2 bursts). Crude membranes were centrifugedat 40,000×g for 15 minutes at 4° C. The membrane pellet was thenresuspended with resuspension buffer and homogenized again with thePolytron tissue disrupter. The protein concentration of the membranesuspension was determined by the method described in Lowry, O. et al.,Journal of Biochemistry 193:265 (1951). All membranes were stored frozenin aliquots at −80° C. or used immediately. Aliquots of prepared hM₅receptor membranes were purchased directly from Perkin Elmer and storedat −80° C. until use.

Radioligand Binding Assay on Muscarinic Receptor Subtypes hM₁, hM₂, hM₃,hM₄ and hM₅

Radioligand binding assays were performed in 96-well microtiter platesin a total assay volume of 1000 μL. CHO cell membranes stably expressingeither the hM₁, hM₂, hM₃, hM₄ or hM₅ muscarinic subtype were diluted inassay buffer to the following specific target protein concentrations(μg/well): 10 μg for hM₁, 10-15 μg for hM₂, 10-20 μg for hM₃, 10-20 μgfor hM₄, and 10-12 μg for hM₅. The membranes were briefly homogenizedusing a Polytron tissue disruptor (10 seconds) prior to assay plateaddition. Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [³H]-NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 40 μM in dilution buffer and then serially diluted 5×with dilution buffer to final concentrations ranging from 400 fM to 4μM. The addition order and volumes to the assay plates were as follows:825 μL assay buffer with 0.1% BSA, 25 μL radioligand, 100 μL dilutedtest compound, and 50 μL membranes. Assay plates were incubated for 6hours at 37° C. Binding reactions were terminated by rapid filtrationover GF/B glass fiber filter plates (Perkin Elmer Inc., Wellesley,Mass.) pre-treated in 0.3% polyethyleneimine. Filter plates were rinsedthree times with wash buffer (10 mM HEPES) to remove unboundradioactivity. Plates were then air dried, and 50 μL Microscint-20liquid scintillation fluid (PerkinElmer Inc., Wellesley, Mass.) wasadded to each well. The plates were then counted in a PerkinElmerTopcount liquid scintillation counter (PerkinElmer Inc., Wellesley,Mass.). Binding data were analyzed by nonlinear regression analysis withthe GraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the one-site competition model. K_(i) values for testcompounds were calculated from observed IC₅₀ values and the K_(D) valueof the radioligand using the Cheng-Prusoff equation (Cheng Y; Prusoff W.H. Biochemical Pharmacology 22(23):3099-108 (1973)). K_(i) values wereconverted to pK_(i) values to determine the geometric mean and 95%confidence intervals. These summary statistics were then converted backto K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher binding affinity for the receptor tested. Exemplary compoundsof the invention that were tested in this assay, were found to have a K,value of less than about 100 nM for the M₃ muscarinic receptor subtypein this assay. More typically, these compounds were found to have K_(i)values of less than about 50 nM, with some compounds having K_(i) valuesof less than about 10 nM or less than about 1.0 nM. For example, thecompounds of Example 1 and Example 2 exhibited a K_(i) value of lessthan about 1.0 nM for the M₃ muscarinic receptor subtype in this assay.

Assay 2 Muscarinic Receptor Functional Potency Assays Blockade ofAgonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of a test compound is determinedby measuring the ability of the test compound to blockoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor.

cAMP assays are performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (NENSMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), according tothe manufacturer's instructions.

Cells are rinsed once with dPBS and lifted with Trypsin-EDTA solution(0.05% trypsin/0.53 mM EDTA) as described in Assay 1. The detached cellsare washed twice by centrifugation at 650×g for five minutes in 50 mLsdPBS. The cell pellet is then re-suspended in 10 mL dPBS, and the cellsare counted with a Coulter Z1 Dual Particle Counter (Beckman Coulter,Fullerton, Calif.). The cells are centrifuged again at 650×g for fiveminutes and re-suspended in stimulation buffer to an assay concentrationof 1.6×10⁶-2.8×10⁶ cells/mL.

The test compound is initially dissolved to a concentration of 400 μM indilution buffer (dPBS supplemented with 1 mg/mL BSA (0.1%)), and thenserially diluted with dilution buffer to final molar concentrationsranging from 100 μM to 0.1 nM. Oxotremorine is diluted in a similarmanner.

To measure oxotremorine inhibition of adenylyl cyclase (AC) activity, 25μL forskolin (25 μM final concentration diluted in dPBS), 25 μL dilutedoxotremorine, and 50 μL cells are added to agonist assay wells. Tomeasure the ability of a test compound to block oxotremorine-inhibitedAC activity, 25 μL forskolin and oxotremorine (25 μM and 5 μM finalconcentrations, respectively, diluted in dPBS) 25 μL diluted testcompound, and 50 μL cells are added to remaining assay wells.

Reactions are incubated for 10 minutes at 37° C. and stopped by additionof 100 μL ice-cold detection buffer. Plates are sealed, incubatedovernight at room temperature and counted the next morning on aPerkinElmer TopCount liquid scintillation counter (PerkinElmer Inc.,Wellesley, Mass.). The amount of cAMP produced (pmol/well) is calculatedbased on the counts observed for the samples and cAMP standards, asdescribed in the manufacturer's user manual. Data are analyzed bynonlinear regression analysis with the GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.) using the non-linearregression, one-site competition equation. The Cheng-Prusoff equation isused to calculate the K_(i), using the EC₅₀ of the oxotremorineconcentration-response curve and the oxotremorine assay concentration asthe K_(D) and [L], respectively. The K values are converted to pK_(i)values to determine the geometric mean and 95% confidence intervals.These summary statistics are then converted back to K_(i) values fordata reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The exemplifiedcompounds of the invention are expected to have a K_(i) value of lessthan about 100 nM for blockade of oxotremorine-inhibition offorskolin-mediated cAMP accumulation in CHO-K1 cells expressing the hM₂receptor. For example, the compound of Example 2 exhibited a K value ofless than about 1.0 nM in this assay.

Blockade of Agonist-Mediated [³⁵S]GTPγS Binding

In a second functional assay, the functional potency of test compoundscan be determined by measuring the ability of the compounds to blockoxotremorine-stimulated [³⁵S]GTPγS binding in CHO-K1 cells expressingthe hM₂ receptor.

At the time of use, frozen membranes are thawed and then diluted inassay buffer with a final target tissue concentration of 5-10 μg proteinper well. The membranes are briefly homogenized using a Polytron PT-2100tissue disrupter and then added to the assay plates.

The EC₉₀ value (effective concentration for 90% maximal response) forstimulation of [³⁵S]GTPγS binding by the agonist oxotremorine isdetermined in each experiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following is added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine (EC₉₀) and guanosine diphosphate (3 μM), 25μL of diluted test compound and 25 μL CHO cell membranes expressing thehM₂ receptor. The assay plates are then incubated at 37° C. for 60minutes. The assay plates are filtered over 1% BSA-pretreated GF/Bfilters using a PerkinElmer 96-well harvester. The plates are rinsedwith ice-cold wash buffer for 3×3 seconds and then air or vacuum dried.Microscint-20 scintillation liquid (50 μL) is added to each well, andeach plate is sealed and radioactivity counted on a topcounter(PerkinElmer). Data are analyzed by nonlinear regression analysis withthe GraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the non-linear regression, one-site competition equation.The Cheng-Prusoff equation is used to calculate the K_(i), using theIC₅₀ values of the concentration-response curve for the test compoundand the oxotremorine concentration in the assay as the K_(D) and [L],ligand concentration, respectively.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The exemplifiedcompounds of the invention are expected to have a K_(i) value of lessthan about 100 nM for blockade of oxotremorine-stimulated [³⁵S]GTPγSbinding in CHO-K1 cells expressing the hM₂ receptor.

Blockade of Agonist-Mediated Calcium Release Via FLIPR Assays

Muscarinic receptor subtypes (M₁, M₃ and M₅ receptors), which couple toG_(q) proteins, activate the phospholipase C (PLC) pathway upon agonistbinding to the receptor. As a result, activated PLC hydrolyzesphosphatyl inositol diphosphate (PIP₂) to diacylglycerol (DAG) andphosphatidyl-1,4,5-triphosphate (IP₃), which in turn generates calciumrelease from intracellular stores, i.e., endoplasmic and sarcoplasmicreticulum. The FLIPR® (Molecular Devices, Inc.) assay capitalizes onthis increase in intracellular calcium by using a calcium sensitive dye(Fluo-4AM, Molecular Probes) that fluoresces when free calcium binds.This fluorescence event is measured in real time by the FLIPR, whichdetects the change in fluorescence from a monolayer of cells cloned withhM₁, hM₃, and cM₅ receptors. Antagonist potency can be determined by theability of antagonists to inhibit agonist-mediated increases inintracellular calcium.

For FLIPR calcium stimulation assays, CHO cells stably expressing thehM₁, hM₃ and cM₅ receptors are seeded into 96-well FLIPR plates thenight before the assay is done. Seeded cells are washed twice byCellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4,2 mM calcium chloride, 2.5 mM probenecid in HBSS without calcium andmagnesium) to remove growth media and leaving 50 L/well of FLIPR buffer.The cells are then incubated with 50 μL/well of 4 M FLUO-4AM (a 2×solution was made) for 40 minutes at 37° C., 5% carbon dioxide.Following the dye incubation period, cells are washed two times withFLIPR buffer, leaving a final volume of 50 μL/well.

To determine antagonist potency, the dose-dependent stimulation ofintracellular Ca²⁺ release for oxotremorine is first determined so thatantagonist potency can later be measured against oxotremorinestimulation at an EC₉₀ concentration. Cells are first incubated withcompound dilution buffer for 20 minutes, followed by agonist addition,which is performed by the FLIPR. An EC₉₀ value for oxotremorine isgenerated according to the method detailed in the FLIPR measurement anddata reduction section below, in conjunction with the formulaEC_(F)=((F/100−F)^1/H)*EC₅₀. An oxotremorine concentration of 3×EC_(F)is prepared in stimulation plates such that an EC₉₀ concentration ofoxotremorine is added to each well in the antagonist inhibition assayplates.

The parameters used for the FLIPR are: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline is determined by measuring thechange in fluorescence for 10 seconds prior to addition of agonist.Following agonist stimulation, the FLIPR continuously measures thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change.

The change of fluorescence is expressed as maximum fluorescence minusbaseline fluorescence for each well. The raw data is analyzed againstthe logarithm of drug concentration by nonlinear regression withGraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) using thebuilt-in model for sigmoidal dose-response. Antagonist K; values aredetermined by Prism using the oxotremorine EC₅₀ value as the K_(D) andthe oxotremorine EC₉₀ for the ligand concentration according to theCheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The exemplifiedcompounds of the invention are expected to have a K_(i) value of lessthan about 100 nM for blockade of agonist-mediated calcium release inCHO cells stably expressing the hM₃ receptor.

Assay 3 Rat Einthoven Assay

This in vivo assay is used to assess the bronchoprotective effects oftest compounds exhibiting muscarinic receptor antagonist activity.

All test compounds are diluted in sterile water and dosed via theinhalation route (IH). The rats (Sprague-Dawley, male, 250-350 g) areexposed to the aerosol generated from an LC Star Nebulizer Set anddriven by a mixture of gases (5% CO₂/95% atmospheric air). Each testcompound solution is nebulized over a 10 minute time period in a pieshaped dosing chamber capable of holding six animals. At predeterminedtime points after inhalation of compound, the Einthoven assay isperformed.

Thirty minutes prior to the start of pulmonary evaluation, the animalsare anesthetized with inactin (thiobutabarbital, 120 mg/kg IP). Thejugular vein is catheterized with saline filled polyethylene catheters(PE-50) and used to infuse the bronchoconstrictor methylcholine (MCh).The trachea is then dissected and cannulated with a 14G needle and usedfor rat ventilation during pulmonary evaluation. Once surgery iscomplete, the animals are ventilated using a piston respirator set at astroke volume of 1 ml/100 g body weight but not exceeding 2.5 ml volume,and at a rate of 90 strokes per minute.

The changes in pressure that occur with each breath are measured.Baseline values are collected for at least 2.5 minutes then the animalsare challenged non-cumulatively with 2-fold incremental increases of MCh(5, 10, 20, 40 and 80 μg/ml). MCh is infused for 2.5 minutes from asyringe pump at a rate of 2 mL/kg/min. The animals are euthanized uponcompletion of the studies.

Changes in ventilation pressure (cm H₂O) in treated animals areexpressed as % inhibition of MCh response relative to control animals.In this assay, a higher % inhibition value indicates that the testcompound has a bronchoprotective effect. Exemplary compounds of theinvention that are tested in this assay at a dose of 100 μg/ml areexpected to exhibit greater than 35% inhibition, some are expected toexhibit greater than 70% inhibition, and some are expected to exhibitgreater than 90% inhibition. For example, the compounds of Example 1 andExample 2 exhibited greater than 35% inhibition in this assay.

1.5 hr ID₅₀ Determination

Standard muscarinic antagonists were evaluated in the rat Einthovenassay 1.5 hours post-dose. The order of potency (ID₅₀s) for the fivestandards tested was determined to be: ipratropium (4.4μg/ml)>tiotropium (6 g/ml)>des-methyl-tiotropium (12μg/ml)>glycopyrrolate (15 μg/ml)>LAS-34237 (24 μg/ml). The potency ofthe test compound is similarly determined at 1.5 hrs post-dose.

6 and 24 hr ID₅₀ Determination

Standards tiotropium and ipratropium were also evaluated 24 hours and/or6 hours post-dose in the rat Einthoven assay. Ipratropium (10 and 30μg/ml) was about 3-fold less potent 6-hours post-dose compared to its1.5 hour potency. The observed loss of activity at this time point (6hours) is consistent with its relatively short duration of action in theclinic. Tiotropium showed a slow onset of effect with peakbronchoprotection being achieved 6-hours post-dose. Its 6 hour and 24hour potency values were not significantly different from each other andwere about 2-fold more potent compared to its 1.5 hour potency. Theonset of action of the test compound, as well as the 6 and 24 hourpotency values, is similarly determined.

Assay 4 Rat Antisialagogue Assay

Rats (Sprague-Dawley, male, 250-350 g) are dosed, anesthetized andcannulated as described for Assay 3. At predetermined time points andafter surgery, animals are placed on their dorsal side at a 20° inclinewith their head in a downward slope. A pre-weighed gauze pad is insertedin the animal's mouth and the muscarinic agonist pilocarpine (PILO) (3mg/kg, IV) is administered. Saliva produced during 10 minutes post-PILOis measured gravimetrically by determining the weight of the gauze padbefore and after PILO. Antisialagogue effects are expressed as %inhibition of salivation relative to control animals.

1, 6 and 24 hr ID₅₀ Determination

The rat antisialagogue assay was developed to assess systemic exposureand calculate the lung selectivity index (LSI) of test compounds. Thestandard, tiotropium, was evaluated in this model at 1, 6, and 24 hourspost-dose. Tiotropium was found to be most potent at inhibitingpilocarpine-induced salivation 6 hours post dose. This finding isconsistent with the peak effects observed in the Einthoven assay.

This model is a modified version of the procedure described in Rechter,“Estimation of anticholinergic drug effects in mice by antagonismagainst pilocarpine-induced salivation” Ata Pharmacol Toxicol 24:243-254(1996). The mean weight of saliva in vehicle-treated animals, at eachpre-treatment time, is calculated and used to compute % inhibition ofsalivation, at the corresponding pre-treatment time, at each dose.

Exemplary compounds of the invention that are tested in this assay areexpected to exhibit ID₅₀ values less than 100 μg/ml (measured at 24hours), with some compounds expected to exhibit an ID₅₀ value less than30 μg/ml, some less than 20 μg/ml, and some less than 15 μg/ml.

The ratio of the anti-sialagogue ID₅₀ to bronchoprotective ID₅₀ is usedto compute the apparent lung selectivity index of the test compound.Generally, compounds having an apparent lung selectivity index greaterthan about 5 are preferred.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

What is claimed is:
 1. A compound having the formula IIf:

where: X⁻ is an anion of a pharmaceutically acceptable acid; R³ isselected from H and —C₀₋₁alkylene-OH; Z is selected from a bond, —O—,—S—, —S(O)—, —SO₂—, —SO₂—NR^(Z1)—, —NR^(Z1)—SO₂—, —C(O)—, —OC(O)—,—C(O)O—, —NR^(Z1)C(O)—, —C(O)NR^(Z1), —NR^(Z2)—C(O)—CR^(Z3)—,—NR^(Z2)—C(S)—NR^(Z3)—, —CH(OH)—, and —C(═N—O—R^(Z4))—; where R^(Z1) isselected from H and —C₁₋₄alkyl; R^(Z2) and R^(Z3) are independentlyselected from H, —C₁₋₄alkyl, and —C₃₋₆cycloalkyl, or R^(Z2) and R^(Z3)are taken together to form —C₂₋₄alkylene- or —C₂₋₃alkenylene-; andR^(Z4) is selected from —C₁₋₄alkyl and benzyl; Q is an aryl orheteroaryl group; wherein the aryl in Q is optionally substituted with 1to 5 R^(Q) groups independently selected from halo, —C₁₋₄alkyl,—C₀₋₄alkylene-OH, cyano, —C₀₋₂alkylene-COOH, —C(O)O—C₁₋₄alkyl,—O—C₁₋₄alkyl, —S—C₁₋₄alkyl, —NH—C(O)—C₁₋₄alkyl, —N-di-C₁₋₄alkyl, and—N⁺(O)O; wherein each alkyl, alkylene, alkenyl, alkenylene, alkynyl, andcycloalkyl group in R³, Z, Q, and R^(Q), is optionally substituted with1 to 5 fluoro atoms; and each —CH₂— group in —(CH₂)₁₋₄— is optionallysubstituted with 1 or 2 substituents independently selected from—C₁₋₂alkyl, —OH, fluoro, and phenyl; or a pharmaceutically acceptablesalt thereof.
 2. The compound of claim 1, wherein Q is a heteroarylgroup selected from thienyl, furanyl, pyrrolyl, pyrazolyl,benzo[1,3]dioxolyl, indolyl, and tetrazolyl.
 3. The compound of claim 1,wherein Z is selected from a bond, —O—, and —C(O)—.
 4. The compound ofclaim 1, wherein Q is phenyl.
 5. The compound of claim 1, wherein Q isunsubstituted.
 6. The compound of claim 4, wherein each R^(Q) isindependently selected from halo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano,—C₀₋₂alkylene-COOH, —C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl, and —N⁺(O)O.
 7. Thecompound of claim 6, wherein Q is substituted with one R^(Q) groupselected from halo, —C₁₋₄-alkyl, —C₀₋₄alkylene-OH, cyano,—C₀₋₂alkylene-COOH, —O—C₁₋₄alkyl, and —N⁺(O)O.
 8. The compound of claim6, wherein Q is substituted with two R^(Q) groups independently selectedfrom halo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, —C(O)O—C₁₋₄alkyl, and—O—C₁₋₄alkyl.
 9. The compound of claim 1, wherein R³ is H; Z is a bond;Q is phenyl, thiophen-2-yl, or thiophen-3-yl; and the phenyl in Q isoptionally substituted with 1 R^(Q) group selected from fluoro and —OH.10. The compound of claim 1, wherein the linker—(CH₂)₀₋₁—N⁺(R⁵R⁶)—(CH₂)₁₋₄—Z— is selected from: —N⁺(CH₃)₂—CH₂—,—N⁺(CH₃)₂—(CH₂)₂—, —N⁺(CH₃)₂—(CH₂)₃—, —N⁺(CH₃)₂—(CH₂)₄—,—N⁺(CH₂)₂—CH₂—CH(phenyl)-, —N⁺(CH₁)₂—CH₂—CH(OH)—, —N⁺(CH₂)₂—CH₂—C(O)—,—N⁺(CH₃)₂—(CH₂)₂—O—, —N⁺(CH₃)₂—(CH₂)₃—O—, —N⁺(CH₃)₂—(CH₂)₄—O—,—CH₂—N⁺(CH₃)₂—CH₂—, —CH₂—N⁺(CH₃)₂—(CH₂)₂—, —CH₂—N⁺(CH₃)₂—(CH₂)₃—,—N⁺(CH₃)(CH₂CH₃)—(CH₂)₂—, —N⁺(CH₂)[(CH₂)₂CH₃]—(CH₂)₂—,—N⁺(CH₂)(CH₂-cyclopropyl)-(CH₂)₂—, —N⁺(CH₃)—(CH₂CH₂OH)—(CH₂)₂—,—N⁺(CH₃)[CH₂C(O)OCH₃]—(CH₂)₂—, and —N⁺(CH₃)—[CH₂C(O)NH₂]—(CH₂)₂—. 11.The compound of claim 1, wherein X is selected from acetate,benzenesulfonate, benzoate, bromide, butyrate, chloride,p-chlorobenzoate, citrate, diphenylacetate, formate, fluoride,o-hydroxybenzoate, p-hydroxybenzoate,1-hydroxynaphthalene-2-carboxylate, 3-hydroxynaphthalene-2-carboxylate,iodide, lactate, malate, maleate, methanesulfonate, nitrate, phosphate,propionate, succinate, sulfate, tartrate, trifluoroacetate, andtriphenylacetate.
 12. The compound of claim 11, wherein X is selectedfrom bromide, iodide and trifluoroacetate.
 13. A pharmaceuticalcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 14. The composition of claim 13, wherein thecompound is in micronized form.
 15. A method for treating a patientsuffering from chronic obstructive pulmonary disease or asthma,comprising administering a therapeutically effective amount of thecompound of claim
 1. 16. The method of claim 15, wherein thetherapeutically effective amount is a bronchodilation-producing amount.